Hydrolases, nucleic acids encoding them and methods for making and using them

ABSTRACT

The invention provides hydrolases, polynucleotides encoding them, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides, e.g., enzymes, having a hydrolase activity, e.g., an esterase, acylase, lipase, phospholipase (e.g., phospholipase A, B, C and D activity, patatin activity, lipid acyl hydrolase (LAH) activity) or protease activity, including thermostable and thermotolerant hydrolase activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The hydrolase activities of the polypeptides and peptides of the invention include esterase activity, lipase activity (hydrolysis of lipids), acidolysis reactions (to replace an esterified fatty acid with a free fatty acid), transesterification reactions (exchange of fatty acids between triglycerides), ester synthesis, ester interchange reactions, phospholipase activity and protease activity (hydrolysis of peptide bonds). The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural and industrial contexts, including the manufacture of cosmetics and nutraceuticals. In another aspect, the polypeptides of the invention are used to synthesize enantiomerically pure chiral products.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.13/619,974, filed Sep. 14, 2012, which is a Divisional of U.S.application Ser. No. 10/547,956, filed Oct. 5, 2006 (issued as U.S. Pat.No. 8,298,799), which is a which is a 371 U.S. National StageApplication of PCT/US2004/007095, filed Mar. 8, 2004, which claimspriority to U.S. Provisional Patent Application Nos. 60/453,450, filedMar. 7, 2003; 60/458,123, filed Mar. 25, 2003; and 60/513,332, filedOct. 21, 2003, the contents of each of which are hereby incorporatedherein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

A Sequence Listing is submitted herewith as an ASCII compliant text filenamed “2919208-177002_Sequence_Listing_ST25.txt”, created on Sep. 23,2015, and having a size of 2,347,596 bytes as permitted under 37 C.F.R.§1.821(c). The material in the aforementioned text file is herebyincorporated by reference in its entirety.

TECHNICAL FIELD

This invention relates to molecular and cellular biology andbiochemistry. In one aspect, the invention provides hydrolases,polynucleotides encoding them, and methods of making and using thesepolynucleotides and polypeptides. In one aspect, the invention isdirected to polypeptides, e.g., enzymes, having a hydrolase activity,e.g., an esterase, acylase, lipase, phospholipase or protease activity,including thermostable and thermotolerant hydrolase activity, andpolynucleotides encoding these enzymes, and making and using thesepolynucleotides and polypeptides. The hydrolase activities of thepolypeptides and peptides of the invention include esterase activity,lipase activity (hydrolysis of lipids), acidolysis reactions (to replacean esterified fatty acid with a free fatty acid), transesterificationreactions (exchange of fatty acids between triglycerides), estersynthesis, ester interchange reactions, phospholipase activity (e.g.,phospholipase A, B, C and D activity, patatin activity, lipid acylhydrolase (LAH) activity) and protease activity (hydrolysis of peptidebonds). The polypeptides of the invention can be used in a variety ofpharmaceutical, agricultural and industrial contexts, including themanufacture of cosmetics and nutraceuticals. In another aspect, thepolypeptides of the invention are used to synthesize enantiomericallypure chiral products.

In one aspect, the polypeptides of the invention are used in thebiocatalytic synthesis of structured lipids (lipids that contain adefined set of fatty acids distributed in a defined manner on theglycerol backbone), including cocoa butter alternatives (CBA), lipidscontaining poly-unsaturated fatty acids (PUFAs), diacylglycerides, e.g.,1,3-diacyl glycerides (DAGs), monoglycerides, e.g., 2-monoglycerides(MAGs) and triacylglycerides (TAGs). In one aspect, the polypeptides ofthe invention are used to modify oils, such as fish, animal andvegetable oils, and lipids, such as poly-unsaturated fatty acids. Thehydrolases of the invention having lipase activity can modify oils byhydrolysis, alcoholysis, esterification, transesterification and/orinteresterification. The methods of the invention can use lipases withdefined regio-specificity or defined chemoselectivity in biocatalyticsynthetic reactions.

Additionally, the polypeptides of the invention can be used in foodprocessing, brewing, bath additives, alcohol production, peptidesynthesis, enantioselectivity, hide preparation in the leather industry,waste management and animal degradation, silver recovery in thephotographic industry, medical treatment, silk degumming, biofilmdegradation, biomass conversion to ethanol, biodefense, antimicrobialagents and disinfectants, personal care and cosmetics, biotech reagents,in increasing starch yield from corn wet milling and pharmaceuticalssuch as digestive aids and anti-inflammatory (anti-phlogistic) agents.

BACKGROUND

The major industrial applications for hydrolases, e.g., esterases,lipases, phospholipases and proteases, include the detergent industry,where they are employed to decompose fatty materials in laundry stainsinto easily removable hydrophilic substances; the food and beverageindustry where they are used in the manufacture of cheese, the ripeningand flavoring of cheese, as antistaling agents for bakery products, andin the production of margarine and other spreads with natural butterflavors; in waste systems; and in the pharmaceutical industry where theyare used as digestive aids.

Oils and fats an important renewable raw material for the chemicalindustry. They are available in large quantities from the processing ofoilseeds from plants like rice bran oil, rapeseed (canola), sunflower,olive, palm or soy. Other sources of valuable oils and fats includefish, restaurant waste, and rendered animal fats. These fats and oilsare a mixture of triglycerides or lipids, i.e. fatty acids (FAs)esterified on a glycerol scaffold. Each oil or fat contains a widevariety of different lipid structures, defined by the FA content andtheir regiochemical distribution on the glycerol backbone. Theseproperties of the individual lipids determine the physical properties ofthe pure triglyceride. Hence, the triglyceride content of a fat or oilto a large extent determines the physical, chemical and biologicalproperties of the oil. The value of lipids increases greatly as afunction of their purity. High purity can be achieved by fractionalchromatography or distillation, separating the desired triglyceride fromthe mixed background of the fat or oil source. However, this is costlyand yields are often limited by the low levels at which the triglycerideoccurs naturally. In addition, the purity of the product is oftencompromised by the presence of many structurally and physically orchemically similar triglycerides in the oil.

An alternative to purifying triglycerides or other lipids from a naturalsource is to synthesize the lipids. The products of such processes arecalled structured lipids because they contain a defined set of fattyacids distributed in a defined manner on the glycerol backbone. Thevalue of lipids also increases greatly by controlling the fatty acidcontent and distribution within the lipid. Lipases can be used to affectsuch control.

Phospholipases are enzymes that hydrolyze the ester bonds ofphospholipids. Corresponding to their importance in the metabolism ofphospholipids, these enzymes are widespread among prokaryotes andeukaryotes. The phospholipases affect the metabolism, construction andreorganization of biological membranes and are involved in signalcascades. Several types of phospholipases are known which differ intheir specificity according to the position of the bond attacked in thephospholipid molecule. Phospholipase A1 (PLA1) removes the 1-positionfatty acid to produce free fatty acid and 1-lyso-2-acylphospholipid.Phospholipase A2 (PLA2) removes the 2-position fatty acid to producefree fatty acid and 1-acyl-2-lysophospholipid. PLA1 and PLA2 enzymes canbe intra- or extra-cellular, membrane-bound or soluble. IntracellularPLA2 is found in almost every mammalian cell. Phospholipase C (PLC)removes the phosphate moiety to produce 1,2 diacylglycerol and phosphobase. Phospholipase D (PLD) produces 1,2-diacylglycerophosphate and basegroup. PLC and PLD are important in cell function and signaling.Patatins are another type of phospholipase thought to work as a PLA.

In general, enzymes, including hydrolases such as esterases, lipases andproteases, are active over a narrow range of environmental conditions(temperature, pH, etc.), and many are highly specific for particularsubstrates. The narrow range of activity for a given enzyme limits itsapplicability and creates a need for a selection of enzymes that (a)have similar activities but are active under different conditions or (b)have different substrates. For instance, an enzyme capable of catalyzinga reaction at 50° C. may be so inefficient at 35° C., that its use atthe lower temperature will not be feasible. For this reason, laundrydetergents generally contain a selection of proteolytic enzymes,allowing the detergent to be used over a broad range of wash temperatureand pH. In view of the specificity of enzymes and the growing use ofhydrolases in industry, research, and medicine, there is an ongoing needin the art for new enzymes and new enzyme inhibitors.

SUMMARY

The invention provides polypeptides, for example, enzymes and catalyticantibodies, having a hydrolase activity, e.g., an esterase, acylase,lipase, phospholipase or protease activity, including thermostable andthermotolerant hydrolase activities, and enantiospecific activities, andpolynucleotides encoding these polypeptides, including vectors, hostcells, transgenic plants and non-human animals, and methods for makingand using these polynucleotides and polypeptides.

The invention provides isolated or recombinant nucleic acids comprisinga nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity to anexemplary nucleic acid of the invention over a region of at least about10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150,175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400,1450, 1500, 1550 or more, residues, wherein the nucleic acid encodes atleast one polypeptide having a hydrolase activity, e.g., an esterase,acylase, lipase, phospholipase or protease activity. The sequenceidentities can be determined by analysis with a sequence comparisonalgorithm or by a visual inspection. Exemplary nucleic acids of theinvention include isolated or recombinant nucleic acids comprising anucleic acid sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ IDNO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179,SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ IDNO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207,SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235,SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ IDNO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263,SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ IDNO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319,SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ IDNO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347,SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353, SEQ ID NO:355, SEQ IDNO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQID NO:367, SEQ ID NO:369, SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:375,SEQ ID NO:377, SEQ ID NO:379, SEQ ID NO:381, SEQ ID NO:383, SEQ IDNO:385, SEQ ID NO:387, SEQ ID NO:389, SEQ ID NO:391, SEQ ID NO:393, SEQID NO:395, SEQ ID NO:397, SEQ ID NO:399, SEQ ID NO:401, SEQ ID NO:403,SEQ ID NO:405, SEQ ID NO:407, SEQ ID NO:409, SEQ ID NO:411, SEQ IDNO:413, SEQ ID NO:415, SEQ ID NO:417, SEQ ID NO:419, SEQ ID NO:421, SEQID NO:423, SEQ ID NO:425, SEQ ID NO:427, SEQ ID NO:429, SEQ ID NO:431,SEQ ID NO:433, SEQ ID NO:435, SEQ ID NO:437, SEQ ID NO:439, SEQ IDNO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ ID NO:449, SEQID NO:451, SEQ ID NO:453, SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459,SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465, SEQ ID NO:467, SEQ IDNO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID NO:475, SEQ ID NO:477, SEQID NO:479, SEQ ID NO:481, SEQ ID NO:483, SEQ ID NO:485, SEQ ID NO:487,SEQ ID NO:489, SEQ ID NO:491, SEQ ID NO:493, SEQ ID NO:495, SEQ IDNO:497, SEQ ID NO:499, SEQ ID NO:501, SEQ ID NO:503, SEQ ID NO:505, SEQID NO:507, SEQ ID NO:509, SEQ ID NO:511, SEQ ID NO:513, SEQ ID NO:515,SEQ ID NO:517, SEQ ID NO:519, SEQ ID NO:521, SEQ ID NO:523, SEQ IDNO:525, SEQ ID NO:527, SEQ ID NO:529, SEQ ID NO:531, SEQ ID NO:533, SEQID NO:535, SEQ ID NO:537, SEQ ID NO:539, SEQ ID NO:541, SEQ ID NO:543,SEQ ID NO:545, SEQ ID NO:547, SEQ ID NO:549, SEQ ID NO:551, SEQ IDNO:553, SEQ ID NO:555, SEQ ID NO:557, SEQ ID NO:559, SEQ ID NO:561, SEQID NO:563, SEQ ID NO:565, SEQ ID NO:567, SEQ ID NO:569, SEQ ID NO:571,SEQ ID NO:573, SEQ ID NO:575, SEQ ID NO:577, SEQ ID NO:579, SEQ IDNO:581, SEQ ID NO:583, SEQ ID NO:585, SEQ ID NO:587, SEQ ID NO:589, SEQID NO:591, SEQ ID NO:593, SEQ ID NO:595, SEQ ID NO:597, SEQ ID NO:599,SEQ ID NO:601, SEQ ID NO:603, SEQ ID NO:605, SEQ ID NO:607, SEQ IDNO:609, SEQ ID NO:611, SEQ ID NO:613, SEQ ID NO:615, SEQ ID NO:617, SEQID NO:619, SEQ ID NO:621, SEQ ID NO:623, SEQ ID NO:625, SEQ ID NO:627,SEQ ID NO:629, SEQ ID NO:631, SEQ ID NO:633, SEQ ID NO:635, SEQ IDNO:637, SEQ ID NO:639, SEQ ID NO:641, SEQ ID NO:643, SEQ ID NO:645, SEQID NO:647, SEQ ID NO:649, SEQ ID NO:651, SEQ ID NO:653, SEQ ID NO:655,SEQ ID NO:657, SEQ ID NO:659, SEQ ID NO:661, SEQ ID NO:663, SEQ IDNO:665, SEQ ID NO:667, SEQ ID NO:669, SEQ ID NO:671, SEQ ID NO:673, SEQID NO:675, SEQ ID NO:677, SEQ ID NO:679, SEQ ID NO:681, SEQ ID NO:683,SEQ ID NO:685, SEQ ID NO:687, SEQ ID NO:689, SEQ ID NO:691, SEQ IDNO:693, SEQ ID NO:695, SEQ ID NO:697, SEQ ID NO:699, SEQ ID NO:701, SEQID NO:703, SEQ ID NO:705, SEQ ID NO:707, SEQ ID NO:709, SEQ ID NO:711,SEQ ID NO:713, SEQ ID NO:715, SEQ ID NO:717, SEQ ID NO:719, SEQ IDNO:721, SEQ ID NO:723, SEQ ID NO:725, SEQ ID NO:727, SEQ ID NO:729, SEQID NO:731, SEQ ID NO:733, SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739,SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, SEQ IDNO:749, SEQ ID NO:751, SEQ ID NO:753, SEQ ID NO:755, SEQ ID NO:757, SEQID NO:759, SEQ ID NO:761, SEQ ID NO:763, SEQ ID NO:765, SEQ ID NO:767,SEQ ID NO:769, SEQ ID NO:771, SEQ ID NO:773, SEQ ID NO:775, SEQ IDNO:777, SEQ ID NO:779, SEQ ID NO:781, SEQ ID NO:783, SEQ ID NO:785, SEQID NO:787, SEQ ID NO:789, SEQ ID NO:791, SEQ ID NO:793, SEQ ID NO:795,SEQ ID NO:797, SEQ ID NO:799, SEQ ID NO:801, SEQ ID NO:803, SEQ IDNO:805, SEQ ID NO:807, SEQ ID NO:809, SEQ ID NO:811, SEQ ID NO:813, SEQID NO:815, SEQ ID NO:817, SEQ ID NO:819, SEQ ID NO:821, SEQ ID NO:823,SEQ ID NO:825, SEQ ID NO:827, SEQ ID NO:829, SEQ ID NO:831, SEQ IDNO:833, SEQ ID NO:835, SEQ ID NO:837, SEQ ID NO:839, SEQ ID NO:841, SEQID NO:843, SEQ ID NO:845, SEQ ID NO:847, SEQ ID NO:849, SEQ ID NO:851,SEQ ID NO:853, SEQ ID NO:855, SEQ ID NO:857, SEQ ID NO:859, SEQ IDNO:861, SEQ ID NO:863, SEQ ID NO:865, SEQ ID NO:867, SEQ ID NO:869, SEQID NO:871, SEQ ID NO:873, SEQ ID NO:875, SEQ ID NO:877, SEQ ID NO:879,SEQ ID NO:881, SEQ ID NO:883, SEQ ID NO:885, SEQ ID NO:887, SEQ IDNO:889, SEQ ID NO:891, SEQ ID NO:893, SEQ ID NO:895, SEQ ID NO:897, SEQID NO:899, SEQ ID NO:901, SEQ ID NO:903, SEQ ID NO:905, SEQ ID NO:907,SEQ ID NO:909, SEQ ID NO:911, SEQ ID NO:913, SEQ ID NO:915, SEQ IDNO:917, SEQ ID NO:919, SEQ ID NO:921, SEQ ID NO:923, SEQ ID NO:925, SEQID NO:927, SEQ ID NO:929, SEQ ID NO:931, SEQ ID NO:933, SEQ ID NO:935,SEQ ID NO:937, SEQ ID NO:939, SEQ ID NO:941, SEQ ID NO:943, SEQ IDNO:945, SEQ ID NO:947, SEQ ID NO:949, SEQ ID NO:951 or SEQ ID NO:953,and subsequences thereof, e.g., at least about 10, 15, 20, 25, 30, 35,40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1450, 1500 or more residues in length, or over thefull length of a gene or transcript.

Additionally, exemplary nucleic acids of the invention include isolated,synthetic or recombinant nucleic acids comprising a nucleic acidsequence as set forth in SEQ ID NO:955, SEQ ID NO:957, SEQ ID NO:959,SEQ ID NO:961, SEQ ID NO:963, SEQ ID NO:965, SEQ ID NO:967, SEQ IDNO:969, SEQ ID NO:971, SEQ ID NO:973, SEQ ID NO:975, SEQ ID NO:977, SEQID NO:979, SEQ ID NO:981, or SEQ ID NO:983, and subsequences thereof.

Exemplary nucleic acids of the invention also include isolated orrecombinant nucleic acids encoding a polypeptide having a sequence asset forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ IDNO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146,SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ IDNO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174,SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ IDNO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202,SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ IDNO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230,SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ IDNO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258,SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ IDNO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286,SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ IDNO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314,SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ IDNO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQID NO:334, SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340, SEQ ID NO:342,SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ ID NO:350, SEQ IDNO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:360, SEQID NO:362, SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368, SEQ ID NO:370,SEQ ID NO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, SEQ IDNO:380, SEQ ID NO:382, SEQ ID NO:384, SEQ ID NO:386, SEQ ID NO:388, SEQID NO:390, SEQ ID NO:392, SEQ ID NO:394, SEQ ID NO:396, SEQ ID NO:398,SEQ ID NO:400, SEQ ID NO:402, SEQ ID NO:404, SEQ ID NO:406, SEQ IDNO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ ID NO:414, SEQ ID NO:416, SEQID NO:418, SEQ ID NO:420, SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426,SEQ ID NO:428, SEQ ID NO:430, SEQ ID NO:432, SEQ ID NO:434, SEQ IDNO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:442, SEQ ID NO:444, SEQID NO:446, SEQ ID NO:448, SEQ ID NO:450, SEQ ID NO:452, SEQ ID NO:454,SEQ ID NO:456, SEQ ID NO:458, SEQ ID NO:460, SEQ ID NO:462, SEQ IDNO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ ID NO:470, SEQ ID NO:472, SEQID NO:474, SEQ ID NO:476, SEQ ID NO:478, SEQ ID NO:480, SEQ ID NO:482,SEQ ID NO:484, SEQ ID NO:486, SEQ ID NO:488, SEQ ID NO:490, SEQ IDNO:492, SEQ ID NO:494, SEQ ID NO:496, SEQ ID NO:498, SEQ ID NO:500, SEQID NO:502, SEQ ID NO:504, SEQ ID NO:506, SEQ ID NO:508, SEQ ID NO:510,SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:516, SEQ ID NO:518, SEQ IDNO:520, SEQ ID NO:522, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQID NO:530, SEQ ID NO:532, SEQ ID NO:534, SEQ ID NO:536, SEQ ID NO:538,SEQ ID NO:540, SEQ ID NO:542, SEQ ID NO:544, SEQ ID NO:546, SEQ IDNO:548, SEQ ID NO:550, SEQ ID NO:552, SEQ ID NO:554, SEQ ID NO:556, SEQID NO:558, SEQ ID NO:560, SEQ ID NO:562, SEQ ID NO:564, SEQ ID NO:566,SEQ ID NO:568, SEQ ID NO:570, SEQ ID NO:572, SEQ ID NO:574, SEQ IDNO:576, SEQ ID NO:578, SEQ ID NO:580, SEQ ID NO:582, SEQ ID NO:584, SEQID NO:586, SEQ ID NO:588, SEQ ID NO:590, SEQ ID NO:592, SEQ ID NO:594,SEQ ID NO:596, SEQ ID NO:598, SEQ ID NO:600, SEQ ID NO:602, SEQ IDNO:604, SEQ ID NO:606, SEQ ID NO:608, SEQ ID NO:610, SEQ ID NO:612, SEQID NO:614, SEQ ID NO:616, SEQ ID NO:618, SEQ ID NO:620, SEQ ID NO:622,SEQ ID NO:624, SEQ ID NO:626, SEQ ID NO:628, SEQ ID NO:630, SEQ IDNO:632, SEQ ID NO:634, SEQ ID NO:636, SEQ ID NO:638, SEQ ID NO:640, SEQID NO:642, SEQ ID NO:644, SEQ ID NO:646, SEQ ID NO:648, SEQ ID NO:650,SEQ ID NO:652, SEQ ID NO:654, SEQ ID NO:656, SEQ ID NO:658, SEQ IDNO:660, SEQ ID NO:662, SEQ ID NO:664, SEQ ID NO:666, SEQ ID NO:668, SEQID NO:670, SEQ ID NO:672, SEQ ID NO:674, SEQ ID NO:676, SEQ ID NO:678,SEQ ID NO:680, SEQ ID NO:682, SEQ ID NO:684, SEQ ID NO:686, SEQ IDNO:688, SEQ ID NO:690, SEQ ID NO:692, SEQ ID NO:694, SEQ ID NO:696, SEQID NO:698, SEQ ID NO:700, SEQ ID NO:702, SEQ ID NO:704, SEQ ID NO:706,SEQ ID NO:708, SEQ ID NO:710, SEQ ID NO:712, SEQ ID NO:714, SEQ IDNO:716, SEQ ID NO:718, SEQ ID NO:720, SEQ ID NO:722, SEQ ID NO:724, SEQID NO:726, SEQ ID NO:728, SEQ ID NO:730, SEQ ID NO:732, SEQ ID NO:734,SEQ ID NO:736, SEQ ID NO:738, SEQ ID NO:740, SEQ ID NO:742, SEQ IDNO:744, SEQ ID NO:746, SEQ ID NO:748, SEQ ID NO:750, SEQ ID NO:752, SEQID NO:754, SEQ ID NO:756, SEQ ID NO:758, SEQ ID NO:760, SEQ ID NO:762,SEQ ID NO:764, SEQ ID NO:766, SEQ ID NO:768, SEQ ID NO:770, SEQ IDNO:772, SEQ ID NO:774, SEQ ID NO:776, SEQ ID NO:778, SEQ ID NO:780, SEQID NO:782, SEQ ID NO:784, SEQ ID NO:786, SEQ ID NO:788, SEQ ID NO:790,SEQ ID NO:792, SEQ ID NO:794, SEQ ID NO:796, SEQ ID NO:798, SEQ IDNO:800, SEQ ID NO:802, SEQ ID NO:804, SEQ ID NO:808, SEQ ID NO:808, SEQID NO:810, SEQ ID NO:812, SEQ ID NO:814, SEQ ID NO:816, SEQ ID NO:818,SEQ ID NO:820, SEQ ID NO:822, SEQ ID NO:824, SEQ ID NO:826, SEQ IDNO:828, SEQ ID NO:830, SEQ ID NO:832, SEQ ID NO:834, SEQ ID NO:836, SEQID NO:838, SEQ ID NO:840, SEQ ID NO:842, SEQ ID NO:844, SEQ ID NO:846,SEQ ID NO:848, SEQ ID NO:850, SEQ ID NO:852, SEQ ID NO:854, SEQ IDNO:856, SEQ ID NO:858, SEQ ID NO:860, SEQ ID NO:862, SEQ ID NO:864, SEQID NO:866, SEQ ID NO:868, SEQ ID NO:870, SEQ ID NO:872, SEQ ID NO:874,SEQ ID NO:876, SEQ ID NO:878, SEQ ID NO:880, SEQ ID NO:882, SEQ IDNO:884, SEQ ID NO:886, SEQ ID NO:888, SEQ ID NO:890, SEQ ID NO:892, SEQID NO:894, SEQ ID NO:896, SEQ ID NO:898, SEQ ID NO:900, SEQ ID NO:902,SEQ ID NO:904, SEQ ID NO:906, SEQ ID NO:908, SEQ ID NO:910, SEQ IDNO:912, SEQ ID NO:914, SEQ ID NO:916, SEQ ID NO:918, SEQ ID NO:920, SEQID NO:922, SEQ ID NO:924, SEQ ID NO:926, SEQ ID NO:928, SEQ ID NO:930,SEQ ID NO:932, SEQ ID NO:934, SEQ ID NO:936, SEQ ID NO:938, SEQ IDNO:940, SEQ ID NO:942, SEQ ID NO:944, SEQ ID NO:946, SEQ ID NO:948, SEQID NO:950, SEQ ID NO:952 or SEQ ID NO:954, and subsequences thereof andvariants thereof. In one aspect, the polypeptide has a hydrolaseactivity, e.g., an esterase, acylase, lipase, phospholipase or proteaseactivity. In one aspect, the hydrolase activity is a regioselectiveand/or chemoselective activity.

Additionally, exemplary nucleic acids of the invention also includeisolated, synthetic or recombinant nucleic acids encoding a polypeptidehaving a sequence as set forth in SEQ ID NO:956, SEQ ID NO:958, SEQ IDNO:960, SEQ ID NO:962, SEQ ID NO:964, SEQ ID NO:966, SEQ ID NO:968, SEQID NO:970, SEQ ID NO:972, SEQ ID NO:974, SEQ ID NO:976, SEQ ID NO:978,SEQ ID NO:980, SEQ ID NO:982, or SEQ ID NO:984, and subsequences thereofand variants thereof.

In one aspect, the invention also provides hydrolase-encoding nucleicacids with a common novelty in that they are derived from mixedcultures. The invention provides hydrolase-encoding nucleic acidsisolated from mixed cultures comprising a nucleic acid of the invention,e.g., a nucleic acid having a sequence at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto an exemplary nucleic acid of the invention over a region of at leastabout 10, 20, 30, 40, 50, 60, 70, 75, 100, 125, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more,residues, wherein the nucleic acid encodes at least one polypeptidehaving a hydrolase activity, and the sequence identities are determinedby analysis with a sequence comparison algorithm or by a visualinspection. In one aspect, the invention provides hydrolase-encodingnucleic acids isolated from mixed cultures comprising a nucleic acid ofthe invention.

In one aspect, the invention also provides hydrolase-encoding nucleicacids with a common novelty in that they are derived from environmentalsources, e.g., mixed environmental sources. In one aspect, the inventionprovides hydrolase-encoding nucleic acids isolated from environmentalsources, e.g., mixed environmental sources, comprising a nucleic acidsequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to an exemplary nucleic acidof the invention over a region of at least about 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more,residues, wherein the nucleic acid encodes at least one polypeptidehaving a hydrolase activity, and the sequence identities are determinedby analysis with a sequence comparison algorithm or by a visualinspection. In one aspect, the invention provides hydrolase-encodingnucleic acids isolated from environmental sources, e.g., mixedenvironmental sources, comprising a nucleic acid of the invention.

In one aspect, the sequence comparison algorithm is a BLAST version2.2.2 algorithm where a filtering setting is set to blastall −p blastp−d “nr pataa”−F F, and all other options are set to default.

Another aspect of the invention is an isolated or recombinant nucleicacid including at least 10 consecutive bases of a nucleic acid sequenceof the invention, sequences substantially identical thereto, and thesequences complementary thereto.

In one aspect, the lipase activity comprises hydrolyzing atriacylglycerol (TAG), a diacylglycerol (DAG) or a monoacylglycerol(MAG). The lipase activity can comprise hydrolyzing a triacylglycerol toa diacylglycerol and a free fatty acid, or, hydrolyzing atriacylglycerol to a monoacylglycerol and free fatty acids, or,hydrolyzing a diacylglycerol to a monoacylglycerol and free fatty acids,or, hydrolyzing a monoacylglycerol to a free fatty acid and a glycerol.The lipase activity can comprise synthesizing a tryacylglycerol from adiacylglycerol or a monoacylglycerol and free fatty acids. The lipaseactivity can comprise synthesizing 1,3-dipalmitoyl-2-oleoylglycerol(POP), 1,3-distearoyl-2-oleoylglycerol (SOS),1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS) or1-oleoyl-2,3-dimyristoylglycerol (OMM), long chain polyunsaturated fattyacids, arachidonic acid, docosahexaenoic acid (DHA) or eicosapentaenoicacid (EPA). The lipase activity can be triacylglycerol (TAG),diacylglycerol (DAG) or monoacylglycerol (MAG) position-specific. Thelipase activity can be Sn2-specific, Sn1- or Sn3-specific. The lipaseactivity can be fatty acid specific. The lipase activity can comprisemodifying oils by hydrolysis, alcoholysis, esterification,transesterification or interesterification. The lipase activity can beregio-specific or chemoselective. The lipase activity can comprisesynthesis of enantiomerically pure chiral products. The lipase activitycan comprise synthesis of umbelliferyl fatty acid (FA) esters.

In one aspect, the isolated or recombinant nucleic acid encodes apolypeptide having a hydrolase activity, e.g., an esterase, acylase,lipase, phospholipase or protease activity, which is thermostable. Thepolypeptide can retain activity under conditions comprising atemperature range of between about 37° C. to about 95° C.; between about55° C. to about 85° C., between about 70° C. to about 95° C., or,between about 90° C. to about 95° C.

In another aspect, the isolated or recombinant nucleic acid encodes apolypeptide having a hydrolase activity, e.g., an esterase, acylase,lipase, phospholipase or protease activity, which is thermotolerant. Thepolypeptide can retain activity after exposure to a temperature in therange from greater than 37° C. to about 95° C. or anywhere in the rangefrom greater than 55° C. to about 85° C. In one aspect, the polypeptideretains activity after exposure to a temperature in the range fromgreater than 90° C. to about 95° C. at pH 4.5.

The invention provides isolated or recombinant nucleic acids comprisinga sequence that hybridizes under stringent conditions to a nucleic acidof the invention, e.g., an exemplary nucleic acid of the inventioncomprising a sequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ IDNO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ IDNO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ IDNO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ IDNO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179,SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ IDNO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207,SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235,SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ IDNO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263,SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ IDNO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319,SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ IDNO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347,SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353, SEQ ID NO:355, SEQ IDNO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQID NO:367, SEQ ID NO:369, SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:375,SEQ ID NO:377, SEQ ID NO:379, SEQ ID NO:381, SEQ ID NO:383, SEQ IDNO:385, SEQ ID NO:387, SEQ ID NO:389, SEQ ID NO:391, SEQ ID NO:393, SEQID NO:395, SEQ ID NO:397, SEQ ID NO:399, SEQ ID NO:401, SEQ ID NO:403,SEQ ID NO:405, SEQ ID NO:407, SEQ ID NO:409, SEQ ID NO:411, SEQ IDNO:413, SEQ ID NO:415, SEQ ID NO:417, SEQ ID NO:419, SEQ ID NO:421, SEQID NO:423, SEQ ID NO:425, SEQ ID NO:427, SEQ ID NO:429, SEQ ID NO:431,SEQ ID NO:433, SEQ ID NO:435, SEQ ID NO:437, SEQ ID NO:439, SEQ IDNO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ ID NO:449, SEQID NO:451, SEQ ID NO:453, SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459,SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465, SEQ ID NO:467, SEQ IDNO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID NO:475, SEQ ID NO:477, SEQID NO:479, SEQ ID NO:481, SEQ ID NO:483, SEQ ID NO:485, SEQ ID NO:487,SEQ ID NO:489, SEQ ID NO:491, SEQ ID NO:493, SEQ ID NO:495, SEQ IDNO:497, SEQ ID NO:499, SEQ ID NO:501, SEQ ID NO:503, SEQ ID NO:505, SEQID NO:507, SEQ ID NO:509, SEQ ID NO:511, SEQ ID NO:513, SEQ ID NO:515,SEQ ID NO:517, SEQ ID NO:519, SEQ ID NO:521, SEQ ID NO:523, SEQ IDNO:525, SEQ ID NO:527, SEQ ID NO:529, SEQ ID NO:531, SEQ ID NO:533, SEQID NO:535, SEQ ID NO:537, SEQ ID NO:539, SEQ ID NO:541, SEQ ID NO:543,SEQ ID NO:545, SEQ ID NO:547, SEQ ID NO:549, SEQ ID NO:551, SEQ IDNO:553, SEQ ID NO:555, SEQ ID NO:557, SEQ ID NO:559, SEQ ID NO:561, SEQID NO:563, SEQ ID NO:565, SEQ ID NO:567, SEQ ID NO:569, SEQ ID NO:571,SEQ ID NO:573, SEQ ID NO:575, SEQ ID NO:577, SEQ ID NO:579, SEQ IDNO:581, SEQ ID NO:583, SEQ ID NO:585, SEQ ID NO:587, SEQ ID NO:589, SEQID NO:591, SEQ ID NO:593, SEQ ID NO:595, SEQ ID NO:597, SEQ ID NO:599,SEQ ID NO:601, SEQ ID NO:603, SEQ ID NO:605, SEQ ID NO:607, SEQ IDNO:609, SEQ ID NO:611, SEQ ID NO:613, SEQ ID NO:615, SEQ ID NO:617, SEQID NO:619, SEQ ID NO:621, SEQ ID NO:623, SEQ ID NO:625, SEQ ID NO:627,SEQ ID NO:629, SEQ ID NO:631, SEQ ID NO:633, SEQ ID NO:635, SEQ IDNO:637, SEQ ID NO:639, SEQ ID NO:641, SEQ ID NO:643, SEQ ID NO:645, SEQID NO:647, SEQ ID NO:649, SEQ ID NO:651, SEQ ID NO:653, SEQ ID NO:655,SEQ ID NO:657, SEQ ID NO:659, SEQ ID NO:661, SEQ ID NO:663, SEQ IDNO:665, SEQ ID NO:667, SEQ ID NO:669, SEQ ID NO:671, SEQ ID NO:673, SEQID NO:675, SEQ ID NO:677, SEQ ID NO:679, SEQ ID NO:681, SEQ ID NO:683,SEQ ID NO:685, SEQ ID NO:687, SEQ ID NO:689, SEQ ID NO:691, SEQ IDNO:693, SEQ ID NO:695, SEQ ID NO:697, SEQ ID NO:699, SEQ ID NO:701, SEQID NO:703, SEQ ID NO:705, SEQ ID NO:707, SEQ ID NO:709, SEQ ID NO:711,SEQ ID NO:713, SEQ ID NO:715, SEQ ID NO:717, SEQ ID NO:719, SEQ IDNO:721, SEQ ID NO:723, SEQ ID NO:725, SEQ ID NO:727, SEQ ID NO:729, SEQID NO:731, SEQ ID NO:733, SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739,SEQ ID NO:741, SEQ ID NO:743, SEQ ID NO:745, SEQ ID NO:747, SEQ IDNO:749, SEQ ID NO:751, SEQ ID NO:753, SEQ ID NO:755, SEQ ID NO:757, SEQID NO:759, SEQ ID NO:761, SEQ ID NO:763, SEQ ID NO:765, SEQ ID NO:767,SEQ ID NO:769, SEQ ID NO:771, SEQ ID NO:773, SEQ ID NO:775, SEQ IDNO:777, SEQ ID NO:779, SEQ ID NO:781, SEQ ID NO:783, SEQ ID NO:785, SEQID NO:787, SEQ ID NO:789, SEQ ID NO:791, SEQ ID NO:793, SEQ ID NO:795,SEQ ID NO:797, SEQ ID NO:799, SEQ ID NO:801, SEQ ID NO:803, SEQ IDNO:805, SEQ ID NO:807, SEQ ID NO:809, SEQ ID NO:811, SEQ ID NO:813, SEQID NO:815, SEQ ID NO:817, SEQ ID NO:819, SEQ ID NO:821, SEQ ID NO:823,SEQ ID NO:825, SEQ ID NO:827, SEQ ID NO:829, SEQ ID NO:831, SEQ IDNO:833, SEQ ID NO:835, SEQ ID NO:837, SEQ ID NO:839, SEQ ID NO:841, SEQID NO:843, SEQ ID NO:845, SEQ ID NO:847, SEQ ID NO:849, SEQ ID NO:851,SEQ ID NO:853, SEQ ID NO:855, SEQ ID NO:857, SEQ ID NO:859, SEQ IDNO:861, SEQ ID NO:863, SEQ ID NO:865, SEQ ID NO:867, SEQ ID NO:869, SEQID NO:871, SEQ ID NO:873, SEQ ID NO:875, SEQ ID NO:877, SEQ ID NO:879,SEQ ID NO:881, SEQ ID NO:883, SEQ ID NO:885, SEQ ID NO:887, SEQ IDNO:889, SEQ ID NO:891, SEQ ID NO:893, SEQ ID NO:895, SEQ ID NO:897, SEQID NO:899, SEQ ID NO:901, SEQ ID NO:903, SEQ ID NO:905, SEQ ID NO:907,SEQ ID NO:909, SEQ ID NO:911, SEQ ID NO:913, SEQ ID NO:915, SEQ IDNO:917, SEQ ID NO:919, SEQ ID NO:921, SEQ ID NO:923, SEQ ID NO:925, SEQID NO:927, SEQ ID NO:929, SEQ ID NO:931, SEQ ID NO:933, SEQ ID NO:935,SEQ ID NO:937, SEQ ID NO:939, SEQ ID NO:941, SEQ ID NO:943, SEQ IDNO:945, SEQ ID NO:947, SEQ ID NO:949, SEQ ID NO:951 or SEQ ID NO:953, orfragments or subsequences thereof. In one aspect, the nucleic acidencodes a polypeptide having a hydrolase activity, e.g., an esterase,acylase, lipase, phospholipase or protease activity. The nucleic acidcan be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1450, 1500 or more residues in length or the fulllength of the gene or transcript. In one aspect, the stringentconditions include a wash step comprising a wash in 0.2×SSC at atemperature of about 65° C. for about 15 minutes.

Additionally, the invention provides isolated, synthetic or recombinantnucleic acids comprising a sequence that hybridizes under stringentconditions to a nucleic acid of the invention, e.g., an exemplarynucleic acid of the invention comprising a sequence as set forth in SEQID NO:955, SEQ ID NO:957, SEQ ID NO:959, SEQ ID NO:961, SEQ ID NO:963,SEQ ID NO:965, SEQ ID NO:967, SEQ ID NO:969, SEQ ID NO:971, SEQ IDNO:973, SEQ ID NO:975, SEQ ID NO:977, SEQ ID NO:979, SEQ ID NO:981, orSEQ ID NO:983, or fragments or subsequences thereof.

The invention provides a nucleic acid probe, e.g., a probe foridentifying a nucleic acid encoding a polypeptide having a hydrolaseactivity, e.g., an esterase, acylase, lipase, phospholipase or proteaseactivity, wherein the probe comprises at least about 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000 or more, consecutive bases of a sequence of the invention, orfragments or subsequences thereof, wherein the probe identifies thenucleic acid by binding or hybridization. The probe can comprise anoligonucleotide comprising at least about 10 to 50, about 20 to 60,about 30 to 70, about 40 to 80, or about 60 to 100 consecutive bases ofa sequence comprising a sequence of the invention, or fragments orsubsequences thereof. The probe can comprise an oligonucleotidecomprising at least about 10 to 50, about 20 to 60, about 30 to 70,about 40 to 80, or about 60 to 100 consecutive bases of a nucleic acidsequence of the invention, or a subsequence thereof.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having a hydrolaseactivity, e.g., an esterase, acylase, lipase, phospholipase or proteaseactivity, wherein the primer pair is capable of amplifying a nucleicacid comprising a sequence of the invention, or fragments orsubsequences thereof. One or each member of the amplification primersequence pair can comprise an oligonucleotide comprising at least about10 to 50 consecutive bases of the sequence.

The invention provides methods of amplifying a nucleic acid encoding apolypeptide having a hydrolase activity, e.g., an esterase, acylase,lipase, phospholipase or protease activity, comprising amplification ofa template nucleic acid with an amplification primer sequence paircapable of amplifying a nucleic acid sequence of the invention, orfragments or subsequences thereof.

The invention provides expression cassettes comprising a nucleic acid ofthe invention or a subsequence thereof. In one aspect, the expressioncassette can comprise the nucleic acid that is operably linked to apromoter. The promoter can be a viral, bacterial, mammalian or plantpromoter. In one aspect, the plant promoter can be a potato, rice, corn,wheat, tobacco or barley promoter. The promoter can be a constitutivepromoter. The constitutive promoter can comprise CaMV35S. In anotheraspect, the promoter can be an inducible promoter. In one aspect, thepromoter can be a tissue-specific promoter or an environmentallyregulated or a developmentally regulated promoter. Thus, the promotercan be, e.g., a seed-specific, a leaf-specific, a root-specific, astem-specific or an abscission-induced promoter. In one aspect, theexpression cassette can further comprise a plant or plant virusexpression vector.

The invention provides cloning vehicles comprising an expressioncassette (e.g., a vector) of the invention or a nucleic acid of theinvention. The cloning vehicle can be a viral vector, a plasmid, aphage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificialchromosome. The viral vector can comprise an adenovirus vector, aretroviral vector or an adeno-associated viral vector. The cloningvehicle can comprise a bacterial artificial chromosome (BAC), a plasmid,a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome(YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cell comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention,or a cloning vehicle of the invention. In one aspect, the transformedcell can be a bacterial cell, a mammalian cell, a fungal cell, a yeastcell, an insect cell or a plant cell. In one aspect, the plant cell canbe a potato, wheat, rice, corn, tobacco or barley cell.

The invention provides transgenic non-human animals comprising a nucleicacid of the invention or an expression cassette (e.g., a vector) of theinvention. In one aspect, the animal is a mouse.

The invention provides transgenic plants comprising a nucleic acid ofthe invention or an expression cassette (e.g., a vector) of theinvention. The transgenic plant can be a corn plant, a potato plant, atomato plant, a wheat plant, an oilseed plant, a rapeseed plant, asoybean plant, a rice plant, a barley plant or a tobacco plant.

The invention provides transgenic seeds comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention.The transgenic seed can be rice, a corn seed, a wheat kernel, anoilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed, asesame seed, a peanut or a tobacco plant seed.

The invention provides an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesmethods of inhibiting the translation of a hydrolase message in a cellcomprising administering to the cell or expressing in the cell anantisense oligonucleotide comprising a nucleic acid sequencecomplementary to or capable of hybridizing under stringent conditions toa nucleic acid of the invention.

The invention provides an isolated or recombinant polypeptide comprisingan amino acid sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more or complete (100%) sequence identity to anexemplary polypeptide or peptide of the invention over a region of atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550 or more residues, or over the full length of thepolypeptide, and the sequence identities are determined by analysis witha sequence comparison algorithm or by a visual inspection. Exemplarypolypeptide or peptide sequences of the invention include SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ IDNO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ IDNO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ IDNO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122,SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ IDNO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQID NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150,SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ IDNO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178,SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ IDNO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206,SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ IDNO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234,SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ IDNO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262,SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ IDNO:272, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290,SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ IDNO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318,SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ IDNO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQID NO:338, SEQ ID NO:340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346,SEQ ID NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ IDNO:356, SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQID NO:366, SEQ ID NO:368, SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374,SEQ ID NO:376, SEQ ID NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ IDNO:384, SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQID NO:394, SEQ ID NO:396, SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402,SEQ ID NO:404, SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ IDNO:412, SEQ ID NO:414, SEQ ID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQID NO:422, SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430,SEQ ID NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ IDNO:440, SEQ ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQID NO:450, SEQ ID NO:452, SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458,SEQ ID NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ IDNO:468, SEQ ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQID NO:478, SEQ ID NO:480, SEQ ID NO:482, SEQ ID NO:484, SEQ ID NO:486,SEQ ID NO:488, SEQ ID NO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ IDNO:496, SEQ ID NO:498, SEQ ID NO:500, SEQ ID NO:502, SEQ ID NO:504, SEQID NO:506, SEQ ID NO:508, SEQ ID NO:510, SEQ ID NO:512, SEQ ID NO:514,SEQ ID NO:516, SEQ ID NO:518, SEQ ID NO:520, SEQ ID NO:522, SEQ IDNO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532, SEQID NO:534, SEQ ID NO:536, SEQ ID NO:538, SEQ ID NO:540, SEQ ID NO:542,SEQ ID NO:544, SEQ ID NO:546, SEQ ID NO:548, SEQ ID NO:550, SEQ IDNO:552, SEQ ID NO:554, SEQ ID NO:556, SEQ ID NO:558, SEQ ID NO:560, SEQID NO:562, SEQ ID NO:564, SEQ ID NO:566, SEQ ID NO:568, SEQ ID NO:570,SEQ ID NO:572, SEQ ID NO:574, SEQ ID NO:576, SEQ ID NO:578, SEQ IDNO:580, SEQ ID NO:582, SEQ ID NO:584, SEQ ID NO:586, SEQ ID NO:588, SEQID NO:590, SEQ ID NO:592, SEQ ID NO:594, SEQ ID NO:596, SEQ ID NO:598,SEQ ID NO:600, SEQ ID NO:602, SEQ ID NO:604, SEQ ID NO:606, SEQ IDNO:608, SEQ ID NO:610, SEQ ID NO:612, SEQ ID NO:614, SEQ ID NO:616, SEQID NO:618, SEQ ID NO:620, SEQ ID NO:622, SEQ ID NO:624, SEQ ID NO:626,SEQ ID NO:628, SEQ ID NO:630, SEQ ID NO:632, SEQ ID NO:634, SEQ IDNO:636, SEQ ID NO:638, SEQ ID NO:640, SEQ ID NO:642, SEQ ID NO:644, SEQID NO:646, SEQ ID NO:648, SEQ ID NO:650, SEQ ID NO:652, SEQ ID NO:654,SEQ ID NO:656, SEQ ID NO:658, SEQ ID NO:660, SEQ ID NO:662, SEQ IDNO:664, SEQ ID NO:666, SEQ ID NO:668, SEQ ID NO:670, SEQ ID NO:672, SEQID NO:674, SEQ ID NO:676, SEQ ID NO:678, SEQ ID NO:680, SEQ ID NO:682,SEQ ID NO:684, SEQ ID NO:686, SEQ ID NO:688, SEQ ID NO:690, SEQ IDNO:692, SEQ ID NO:694, SEQ ID NO:696, SEQ ID NO:698, SEQ ID NO:700, SEQID NO:702, SEQ ID NO:704, SEQ ID NO:706, SEQ ID NO:708, SEQ ID NO:710,SEQ ID NO:712, SEQ ID NO:714, SEQ ID NO:716, SEQ ID NO:718, SEQ IDNO:720, SEQ ID NO:722, SEQ ID NO:724, SEQ ID NO:726, SEQ ID NO:728, SEQID NO:730, SEQ ID NO:732, SEQ ID NO:734, SEQ ID NO:736, SEQ ID NO:738,SEQ ID NO:740, SEQ ID NO:742, SEQ ID NO:744, SEQ ID NO:746, SEQ IDNO:748, SEQ ID NO:750, SEQ ID NO:752, SEQ ID NO:754, SEQ ID NO:756, SEQID NO:758, SEQ ID NO:760, SEQ ID NO:762, SEQ ID NO:764, SEQ ID NO:766,SEQ ID NO:768, SEQ ID NO:770, SEQ ID NO:772, SEQ ID NO:774, SEQ IDNO:776, SEQ ID NO:778, SEQ ID NO:780, SEQ ID NO:782, SEQ ID NO:784, SEQID NO:786, SEQ ID NO:788, SEQ ID NO:790, SEQ ID NO:792, SEQ ID NO:794,SEQ ID NO:796, SEQ ID NO:798, SEQ ID NO:800, SEQ ID NO:802, SEQ IDNO:804, SEQ ID NO:808, SEQ ID NO:808, SEQ ID NO:810, SEQ ID NO:812, SEQID NO:814, SEQ ID NO:816, SEQ ID NO:818, SEQ ID NO:820, SEQ ID NO:822,SEQ ID NO:824, SEQ ID NO:826, SEQ ID NO:828, SEQ ID NO:830, SEQ IDNO:832, SEQ ID NO:834, SEQ ID NO:836, SEQ ID NO:838, SEQ ID NO:840, SEQID NO:842, SEQ ID NO:844, SEQ ID NO:846, SEQ ID NO:848, SEQ ID NO:850,SEQ ID NO:852, SEQ ID NO:854, SEQ ID NO:856, SEQ ID NO:858, SEQ IDNO:860, SEQ ID NO:862, SEQ ID NO:864, SEQ ID NO:866, SEQ ID NO:868, SEQID NO:870, SEQ ID NO:872, SEQ ID NO:874, SEQ ID NO:876, SEQ ID NO:878,SEQ ID NO:880, SEQ ID NO:882, SEQ ID NO:884, SEQ ID NO:886, SEQ IDNO:888, SEQ ID NO:890, SEQ ID NO:892, SEQ ID NO:894, SEQ ID NO:896, SEQID NO:898, SEQ ID NO:900, SEQ ID NO:902, SEQ ID NO:904, SEQ ID NO:906,SEQ ID NO:908, SEQ ID NO:910, SEQ ID NO:912, SEQ ID NO:914, SEQ IDNO:916, SEQ ID NO:918, SEQ ID NO:920, SEQ ID NO:922, SEQ ID NO:924, SEQID NO:926, SEQ ID NO:928, SEQ ID NO:930, SEQ ID NO:932, SEQ ID NO:934,SEQ ID NO:936, SEQ ID NO:938, SEQ ID NO:940, SEQ ID NO:942, SEQ IDNO:944, SEQ ID NO:946, SEQ ID NO:948, SEQ ID NO:950, SEQ ID NO:952 andSEQ ID NO:954, and subsequences thereof and variants thereof, e.g., atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 or moreresidues in length, or over the full length of an enzyme. Exemplarypolypeptide or peptide sequences of the invention include sequenceencoded by a nucleic acid of the invention. Exemplary polypeptide orpeptide sequences of the invention include polypeptides or peptidesspecifically bound by an antibody of the invention. In one aspect, apolypeptide of the invention has at least one hydrolase activity, e.g.,an esterase, acylase, lipase, phospholipase or protease activity. In oneaspect, the activity is a regioselective and/or chemoselective activity.

Additionally, exemplary polypeptide or peptide sequences of theinvention include SEQ ID NO:956, SEQ ID NO:958, SEQ ID NO:960, SEQ IDNO:962, SEQ ID NO:964, SEQ ID NO:966, SEQ ID NO:968, SEQ ID NO:970, SEQID NO:972, SEQ ID NO:974, SEQ ID NO:976, SEQ ID NO:978, SEQ ID NO:980,SEQ ID NO:982, or SEQ ID NO:984, and subsequences thereof and variantsthereat: e.g., at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400,1450, 1500 or more residues in length, or over the full length of anenzyme.

Another aspect of the invention is an isolated or recombinantpolypeptide or peptide including at least 10 consecutive bases of apolypeptide or peptide sequence of the invention, sequencessubstantially identical thereto, and the sequences complementarythereto.

In one aspect, the lipase activity comprises hydrolyzing atriacylglycerol (TAG), a diacylglycerol (DAG) or a monoacylglycerol(MAG). The lipase activity can comprise hydrolyzing a triacylglycerol toa diacylglycerol and a free fatty acid, or, hydrolyzing atriacylglycerol to a monoacylglycerol and free fatty acids, or,hydrolyzing a diacylglycerol to a monoacylglycerol and free fatty acids,or, hydrolyzing a monoacylglycerol to a free fatty acid and a glycerol.The lipase activity can comprise synthesizing a tryacylglycerol from adiacylglycerol or a monoacylglycerol and free fatty acids. The lipaseactivity can comprise synthesizing 1,3-dipalmitoyl-2-oleoylglycerol(POP), 1,3-distearoyl-2-oleoylglycerol (SOS),1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS) or1-oleoyl-2,3-dimyristoylglycerol (OMM), long chain polyunsaturated fattyacids, arachidonic acid, docosahexaenoic acid (DHA) or eicosapentaenoicacid (EPA). The lipase activity can be triacylglycerol (TAG),diacylglycerol (DAG) or monoacylglycerol (MAG) position-specific. Thelipase activity can be Sn2-specific, Sn1- or Sn3-specific. The lipaseactivity can be fatty acid specific. The lipase activity can comprisemodifying oils by hydrolysis, alcoholysis, esterification,transesterification or interesterification. The lipase activity can beregio-specific or chemoselective. The lipase activity can comprisesynthesis of enantiomerically pure chiral products. The lipase activitycan comprise synthesis of umbelliferyl fatty acid (FA) esters.

In one aspect, the hydrolase activity can be thermostable. Thepolypeptide can retain a hydrolase activity under conditions comprisinga temperature range of between about 37° C. to about 95° C., betweenabout 55° C. to about 85° C., between about 70° C. to about 95° C., orbetween about 90° C. to about 95° C. In another aspect, the hydrolaseactivity can be thermotolerant. The polypeptide can retain a hydrolaseactivity after exposure to a temperature in the range from greater than37° C. to about 95° C., or in the range from greater than 55° C. toabout 85° C. In one aspect, the polypeptide can retain a hydrolaseactivity after exposure to a temperature in the range from greater than90° C. to about 95° C. at pH 4.5.

In one aspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention that lacks a signal sequence. In oneaspect, the isolated or recombinant polypeptide can comprise thepolypeptide of the invention comprising a heterologous signal sequence,such as a heterologous hydrolase or non-hydrolase signal sequence. Inone aspect, the invention provides chimeric proteins comprising a firstdomain comprising a signal sequence of the invention and at least asecond domain. The protein can be a fusion protein. The second domaincan comprise an enzyme. The enzyme can be a hydrolase (e.g., a hydrolaseof the invention, or, another hydrolase).

In one aspect, the hydrolase activity comprises a specific activity atabout 37° C. in the range from about 100 to about 1000 units permilligram of protein. In another aspect, the hydrolase activitycomprises a specific activity from about 500 to about 750 units permilligram of protein. Alternatively, the hydrolase activity comprises aspecific activity at 37° C. in the range from about 500 to about 1200units per milligram of protein. In one aspect, the hydrolase activitycomprises a specific activity at 37° C. in the range from about 750 toabout 1000 units per milligram of protein. In another aspect, thethermotolerance comprises retention of at least half of the specificactivity of the hydrolase at 37° C. after being heated to the elevatedtemperature. Alternatively, the thermotolerance can comprise retentionof specific activity at 37° C. in the range from about 500 to about 1200units per milligram of protein after being heated to the elevatedtemperature.

The invention provides the isolated or recombinant polypeptide of theinvention, wherein the polypeptide comprises at least one glycosylationsite. In one aspect, glycosylation can be an N-linked glycosylation. Inone aspect, the polypeptide can be glycosylated after being expressed ina P. pastoris or a S. pombe.

In one aspect, the polypeptide can retain a hydrolase activity underconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4.In another aspect, the polypeptide can retain a hydrolase activity underconditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5,pH 10, pH 10.5 or pH 11.

The invention provides protein preparations comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, asolid or a gel.

The invention provides heterodimers comprising a polypeptide of theinvention and a second domain. In one aspect, the second domain can be apolypeptide and the heterodimer can be a fusion protein. In one aspect,the second domain can be an epitope or a tag. In one aspect, theinvention provides homodimers comprising a polypeptide of the invention.

The invention provides immobilized polypeptides having a hydrolaseactivity, wherein the polypeptide comprises a polypeptide of theinvention, a polypeptide encoded by a nucleic acid of the invention, ora polypeptide comprising a polypeptide of the invention and a seconddomain. In one aspect, the polypeptide can be immobilized on a cell, ametal, a resin, a polymer, a ceramic, a glass, a microelectrode, agraphitic particle, a bead, a gel, a plate, an array or a capillarytube.

The invention provides arrays comprising an immobilized nucleic acid ofthe invention. The invention provides arrays comprising an antibody ofthe invention.

The invention provides isolated or recombinant antibodies thatspecifically bind to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention. The antibody can be amonoclonal or a polyclonal antibody. The invention provides hybridomascomprising an antibody of the invention, e.g., an antibody thatspecifically binds to a polypeptide of the invention or to a polypeptideencoded by a nucleic acid of the invention.

The invention provides food supplements for an animal comprising apolypeptide of the invention, e.g., a polypeptide encoded by the nucleicacid of the invention. In one aspect, the polypeptide in the foodsupplement can be glycosylated. The invention provides edible enzymedelivery matrices comprising a polypeptide of the invention, e.g., apolypeptide encoded by the nucleic acid of the invention. In one aspect,the delivery matrix comprises a pellet. In one aspect, the polypeptidecan be glycosylated. In one aspect, the hydrolase activity isthermotolerant. In another aspect, the hydrolase activity isthermostable.

The invention provides method of isolating or identifying a polypeptidehaving a hydrolase activity comprising the steps of: (a) providing anantibody of the invention; (b) providing a sample comprisingpolypeptides; and (c) contacting the sample of step (b) with theantibody of step (a) under conditions wherein the antibody canspecifically bind to the polypeptide, thereby isolating or identifying apolypeptide having a hydrolase activity.

The invention provides methods of making an anti-hydrolase antibodycomprising administering to a non-human animal a nucleic acid of theinvention or a polypeptide of the invention or subsequences thereof inan amount sufficient to generate a humoral immune response, therebymaking an anti-hydrolase antibody. The invention provides methods ofmaking an anti-hydrolase immune comprising administering to a non-humananimal a nucleic acid of the invention or a polypeptide of the inventionor subsequences thereof in an amount sufficient to generate an immuneresponse.

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid of the inventionoperably linked to a promoter; and (b) expressing the nucleic acid ofstep (a) under conditions that allow expression of the polypeptide,thereby producing a recombinant polypeptide. In one aspect, the methodcan further comprise transforming a host cell with the nucleic acid ofstep (a) followed by expressing the nucleic acid of step (a), therebyproducing a recombinant polypeptide in a transformed cell.

The invention provides methods for identifying a polypeptide having ahydrolase activity comprising the following steps: (a) providing apolypeptide of the invention; or a polypeptide encoded by a nucleic acidof the invention; (b) providing a hydrolase substrate; and (c)contacting the polypeptide or a fragment or variant thereof of step (a)with the substrate of step (b) and detecting a decrease in the amount ofsubstrate or an increase in the amount of a reaction product, wherein adecrease in the amount of the substrate or an increase in the amount ofthe reaction product detects a polypeptide having a hydrolase activity.In alternative aspects, the substrate can be a poly-unsaturated fattyacid (PUFA), a diacylglyceride, e.g., a 1,3-diacyl glyceride (DAG), amonoglyceride, e.g., 2-monoglyceride (MAG) or a triacylglyceride (TAG).

The invention provides methods for identifying a hydrolase substratecomprising the following steps: (a) providing a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid of the invention;(b) providing a test substrate; and (c) contacting the polypeptide ofstep (a) with the test substrate of step (b) and detecting a decrease inthe amount of substrate or an increase in the amount of reactionproduct, wherein a decrease in the amount of the substrate or anincrease in the amount of a reaction product identifies the testsubstrate as a hydrolase substrate.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising the following steps: (a)expressing a nucleic acid or a vector comprising the nucleic acid underconditions permissive for translation of the nucleic acid to apolypeptide, wherein the nucleic acid comprises a nucleic acid of theinvention, or, providing a polypeptide of the invention; (b) providing atest compound; (c) contacting the polypeptide with the test compound;and (d) determining whether the test compound of step (b) specificallybinds to the polypeptide.

The invention provides methods for identifying a modulator of ahydrolase activity comprising the following steps: (a) providing apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention; (b) providing a test compound; (c) contacting thepolypeptide of step (a) with the test compound of step (b) and measuringan activity of the hydrolase, wherein a change in the hydrolase activitymeasured in the presence of the test compound compared to the activityin the absence of the test compound provides a determination that thetest compound modulates the hydrolase activity. In one aspect, thehydrolase activity can be measured by providing a hydrolase substrateand detecting a decrease in the amount of the substrate or an increasein the amount of a reaction product, or, an increase in the amount ofthe substrate or a decrease in the amount of a reaction product. Adecrease in the amount of the substrate or an increase in the amount ofthe reaction product with the test compound as compared to the amount ofsubstrate or reaction product without the test compound identifies thetest compound as an activator of hydrolase activity. An increase in theamount of the substrate or a decrease in the amount of the reactionproduct with the test compound as compared to the amount of substrate orreaction product without the test compound identifies the test compoundas an inhibitor of hydrolase activity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence or a nucleic acid sequence of the invention(e.g., a polypeptide encoded by a nucleic acid of the invention). In oneaspect, the computer system can further comprise a sequence comparisonalgorithm and a data storage device having at least one referencesequence stored thereon. In another aspect, the sequence comparisonalgorithm comprises a computer program that indicates polymorphisms. Inone aspect, the computer system can further comprise an identifier thatidentifies one or more features in said sequence. The invention providescomputer readable media having stored thereon a polypeptide sequence ora nucleic acid sequence of the invention. The invention provides methodsfor identifying a feature in a sequence comprising the steps of: (a)reading the sequence using a computer program which identifies one ormore features in a sequence, wherein the sequence comprises apolypeptide sequence or a nucleic acid sequence of the invention; and(b) identifying one or more features in the sequence with the computerprogram. The invention provides methods for comparing a first sequenceto a second sequence comprising the steps of: (a) reading the firstsequence and the second sequence through use of a computer program whichcompares sequences, wherein the first sequence comprises a polypeptidesequence or a nucleic acid sequence of the invention; and (b)determining differences between the first sequence and the secondsequence with the computer program. The step of determining differencesbetween the first sequence and the second sequence can further comprisethe step of identifying polymorphisms. In one aspect, the method canfurther comprise an identifier that identifies one or more features in asequence. In another aspect, the method can comprise reading the firstsequence using a computer program and identifying one or more featuresin the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having a hydrolase activity from anenvironmental sample comprising the steps of: (a) providing anamplification primer sequence pair for amplifying a nucleic acidencoding a polypeptide having a hydrolase activity, wherein the primerpair is capable of amplifying a nucleic acid of the invention; (b)isolating a nucleic acid from the environmental sample or treating theenvironmental sample such that nucleic acid in the sample is accessiblefor hybridization to the amplification primer pair; and, (c) combiningthe nucleic acid of step (b) with the amplification primer pair of step(a) and amplifying nucleic acid from the environmental sample, therebyisolating or recovering a nucleic acid encoding a polypeptide having ahydrolase activity from an environmental sample. One or each member ofthe amplification primer sequence pair can comprise an oligonucleotidecomprising at least about 10 to 50 consecutive bases of a sequence ofthe invention.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having a hydrolase activity from anenvironmental sample comprising the steps of: (a) providing apolynucleotide probe comprising a nucleic acid of the invention or asubsequence thereof; (b) isolating a nucleic acid from the environmentalsample or treating the environmental sample such that nucleic acid inthe sample is accessible for hybridization to a polynucleotide probe ofstep (a); (c) combining the isolated nucleic acid or the treatedenvironmental sample of step (b) with the polynucleotide probe of step(a); and (d) isolating a nucleic acid that specifically hybridizes withthe polynucleotide probe of step (a), thereby isolating or recovering anucleic acid encoding a polypeptide having a hydrolase activity from anenvironmental sample. The environmental sample can comprise a watersample, a liquid sample, a soil sample, an air sample or a biologicalsample. In one aspect, the biological sample can be derived from abacterial cell, a protozoan cell, an insect cell, a yeast cell, a plantcell, a fungal cell or a mammalian cell.

The invention provides methods of generating a variant of a nucleic acidencoding a polypeptide having a hydrolase activity comprising the stepsof: (a) providing a template nucleic acid comprising a nucleic acid ofthe invention; and (b) modifying, deleting or adding one or morenucleotides in the template sequence, or a combination thereof, togenerate a variant of the template nucleic acid. In one aspect, themethod can further comprise expressing the variant nucleic acid togenerate a variant hydrolase polypeptide. The modifications, additionsor deletions can be introduced by a method comprising error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM),synthetic ligation reassembly (SLR) or a combination thereof. In anotheraspect, the modifications, additions or deletions are introduced by amethod comprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

In one aspect, the method can be iteratively repeated until a hydrolasehaving an altered or different activity or an altered or differentstability from that of a polypeptide encoded by the template nucleicacid is produced. In one aspect, the variant hydrolase polypeptide isthermotolerant, and retains some activity after being exposed to anelevated temperature. In another aspect, the variant hydrolasepolypeptide has increased glycosylation as compared to the hydrolaseencoded by a template nucleic acid. Alternatively, the variant hydrolasepolypeptide has a hydrolase activity under a high temperature, whereinthe hydrolase encoded by the template nucleic acid is not active underthe high temperature. In one aspect, the method can be iterativelyrepeated until a hydrolase coding sequence having an altered codon usagefrom that of the template nucleic acid is produced. In another aspect,the method can be iteratively repeated until a hydrolase gene havinghigher or lower level of message expression or stability from that ofthe template nucleic acid is produced.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a hydrolase activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention encoding a polypeptidehaving a hydrolase activity; and, (b) identifying a non-preferred or aless preferred codon in the nucleic acid of step (a) and replacing itwith a preferred or neutrally used codon encoding the same amino acid asthe replaced codon, wherein a preferred codon is a codonover-represented in coding sequences in genes in the host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to increase its expression in a host cell.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a hydrolase activity; the methodcomprising the following steps: (a) providing a nucleic acid of theinvention; and, (b) identifying a codon in the nucleic acid of step (a)and replacing it with a different codon encoding the same amino acid asthe replaced codon, thereby modifying codons in a nucleic acid encodinga hydrolase.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a hydrolase activity to increase itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention encoding a hydrolasepolypeptide; and, (b) identifying a non-preferred or a less preferredcodon in the nucleic acid of step (a) and replacing it with a preferredor neutrally used codon encoding the same amino acid as the replacedcodon, wherein a preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell, thereby modifying the nucleic acid to increaseits expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding a polypeptide having a hydrolase activity to decrease itsexpression in a host cell, the method comprising the following steps:(a) providing a nucleic acid of the invention; and (b) identifying atleast one preferred codon in the nucleic acid of step (a) and replacingit with a non-preferred or less preferred codon encoding the same aminoacid as the replaced codon, wherein a preferred codon is a codonover-represented in coding sequences in genes in a host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to decrease its expression in a host cell. In one aspect,the host cell can be a bacterial cell, a fungal cell, an insect cell, ayeast cell, a plant cell or a mammalian cell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified hydrolase active sites or substratebinding sites, wherein the modified active sites or substrate bindingsites are derived from a first nucleic acid comprising a sequenceencoding a first active site or a first substrate binding site themethod comprising the following steps: (a) providing a first nucleicacid encoding a first active site or first substrate binding site,wherein the first nucleic acid sequence comprises a sequence thathybridizes under stringent conditions to a nucleic acid of theinvention, and the nucleic acid encodes a hydrolase active site or ahydrolase substrate binding site; (b) providing a set of mutagenicoligonucleotides that encode naturally-occurring amino acid variants ata plurality of targeted codons in the first nucleic acid; and, (c) usingthe set of mutagenic oligonucleotides to generate a set of activesite-encoding or substrate binding site-encoding variant nucleic acidsencoding a range of amino acid variations at each amino acid codon thatwas mutagenized, thereby producing a library of nucleic acids encoding aplurality of modified hydrolase active sites or substrate binding sites.In one aspect, the method comprises mutagenizing the first nucleic acidof step (a) by a method comprising an optimized directed evolutionsystem, gene site-saturation mutagenesis (GSSM), synthetic ligationreassembly (SLR), error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR)and a combination thereof. In another aspect, the method comprisesmutagenizing the first nucleic acid of step (a) or variants by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

The invention provides methods for making a small molecule comprisingthe following steps: (a) providing a plurality of biosynthetic enzymescapable of synthesizing or modifying a small molecule, wherein one ofthe enzymes comprises a hydrolase enzyme encoded by a nucleic acid ofthe invention; (b) providing a substrate for at least one of the enzymesof step (a); and (c) reacting the substrate of step (b) with the enzymesunder conditions that facilitate a plurality of biocatalytic reactionsto generate a small molecule by a series of biocatalytic reactions. Theinvention provides methods for modifying a small molecule comprising thefollowing steps: (a) providing a hydrolase enzyme, wherein the enzymecomprises a polypeptide of the invention, or, a polypeptide encoded by anucleic acid of the invention, or a subsequence thereof; (b) providing asmall molecule; and (c) reacting the enzyme of step (a) with the smallmolecule of step (b) under conditions that facilitate an enzymaticreaction catalyzed by the hydrolase enzyme, thereby modifying a smallmolecule by a hydrolase enzymatic reaction. In one aspect, the methodcan comprise a plurality of small molecule substrates for the enzyme ofstep (a), thereby generating a library of modified small moleculesproduced by at least one enzymatic reaction catalyzed by the hydrolaseenzyme. In one aspect, the method can comprise a plurality of additionalenzymes under conditions that facilitate a plurality of biocatalyticreactions by the enzymes to form a library of modified small moleculesproduced by the plurality of enzymatic reactions. In another aspect, themethod can further comprise the step of testing the library to determineif a particular modified small molecule which exhibits a desiredactivity is present within the library. The step of testing the librarycan further comprise the steps of systematically eliminating all but oneof the biocatalytic reactions used to produce a portion of the pluralityof the modified small molecules within the library by testing theportion of the modified small molecule for the presence or absence ofthe particular modified small molecule with a desired activity, andidentifying at least one specific biocatalytic reaction that producesthe particular modified small molecule of desired activity.

The invention provides methods for determining a functional fragment ofa hydrolase enzyme comprising the steps of: (a) providing a hydrolaseenzyme, wherein the enzyme comprises a polypeptide of the invention, ora polypeptide encoded by a nucleic acid of the invention, or asubsequence thereof; and (b) deleting a plurality of amino acid residuesfrom the sequence of step (a) and testing the remaining subsequence fora hydrolase activity, thereby determining a functional fragment of ahydrolase enzyme. In one aspect, the hydrolase activity is measured byproviding a hydrolase substrate and detecting a decrease in the amountof the substrate or an increase in the amount of a reaction product.

The invention provides methods for whole cell engineering of new ormodified phenotypes by using real-time metabolic flux analysis, themethod comprising the following steps: (a) making a modified cell bymodifying the genetic composition of a cell, wherein the geneticcomposition is modified by addition to the cell of a nucleic acid of theinvention; (b) culturing the modified cell to generate a plurality ofmodified cells; (c) measuring at least one metabolic parameter of thecell by monitoring the cell culture of step (b) in real time; and, (d)analyzing the data of step (c) to determine if the measured parameterdiffers from a comparable measurement in an unmodified cell undersimilar conditions, thereby identifying an engineered phenotype in thecell using real-time metabolic flux analysis. In one aspect, the geneticcomposition of the cell can be modified by a method comprising deletionof a sequence or modification of a sequence in the cell, or, knockingout the expression of a gene. In one aspect, the method can furthercomprise selecting a cell comprising a newly engineered phenotype. Inanother aspect, the method can comprise culturing the selected cell,thereby generating a new cell strain comprising a newly engineeredphenotype.

The invention provides methods of increasing thermotolerance orthermostability of a hydrolase polypeptide, the method comprisingglycosylating a hydrolase polypeptide, wherein the polypeptide comprisesat least thirty contiguous amino acids of a polypeptide of theinvention; or a polypeptide encoded by a nucleic acid sequence of theinvention, thereby increasing the thermotolerance or thermostability ofthe hydrolase polypeptide. In one aspect, the hydrolase specificactivity can be thermostable or thermotolerant at a temperature in therange from greater than about 37° C. to about 95° C.

The invention provides methods for overexpres sing a recombinanthydrolase polypeptide in a cell comprising expressing a vectorcomprising a nucleic acid comprising a nucleic acid of the invention ora nucleic acid sequence of the invention, wherein the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by visual inspection, wherein overexpression is effected byuse of a high activity promoter, a dicistronic vector or by geneamplification of the vector.

The invention provides detergent compositions comprising a polypeptideof the invention or a polypeptide encoded by a nucleic acid of theinvention, wherein the polypeptide comprises a hydrolase activity, e.g.,an esterase, acylase, lipase, phospholipase or protease activity. In oneaspect, the hydrolase can be a nonsurface-active hydrolase. In anotheraspect, the hydrolase can be a surface-active hydrolase.

The invention provides methods for washing an object comprising thefollowing steps: (a) providing a composition comprising a polypeptidehaving a hydrolase activity, e.g., an esterase, acylase, lipase,phospholipase or protease activity, wherein the polypeptide comprises: apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention; (b) providing an object; and (c) contacting thepolypeptide of step (a) and the object of step (b) under conditionswherein the composition can wash the object.

The invention provides methods of making a transgenic plant comprisingthe following steps: (a) introducing a heterologous nucleic acidsequence into the cell, wherein the heterologous nucleic sequencecomprises a nucleic acid sequence of the invention, thereby producing atransformed plant cell; and (b) producing a transgenic plant from thetransformed cell. In one aspect, the step (a) can further compriseintroducing the heterologous nucleic acid sequence by electroporation ormicroinjection of plant cell protoplasts. In another aspect, the step(a) can further comprise introducing the heterologous nucleic acidsequence directly to plant tissue by DNA particle bombardment.Alternatively, the step (a) can further comprise introducing theheterologous nucleic acid sequence into the plant cell DNA using anAgrobacterium tumefaciens host. In one aspect, the plant cell can be apotato, corn, rice, wheat, tobacco, or barley cell.

The invention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising the following steps: (a)transforming the plant cell with a heterologous nucleic acid sequenceoperably linked to a promoter, wherein the heterologous nucleic sequencecomprises a nucleic acid of the invention; (b) growing the plant underconditions wherein the heterologous nucleic acids sequence is expressedin the plant cell.

The invention provides signal sequences comprising or consisting of apeptide having a subsequence of a polypeptide of the invention. Theinvention provides a chimeric protein comprising a first domaincomprising a signal sequence of the invention and at least a seconddomain. The protein can be a fusion protein. The second domain cancomprise an enzyme. The enzyme can be a hydrolase.

The invention provides method for biocatalytic synthesis of a structuredlipid comprising the following steps: (a) providing a hydrolase of theinvention; (b) providing a composition comprising a triacylglyceride(TAG); (c) contacting the polypeptide of step (a) with the compositionof step (b) under conditions wherein the polypeptide hydrolyzes an acylresidue at the Sn2 position of the triacylglyceride (TAG), therebyproducing a 1,3-diacylglyceride (DAG); (d) providing an R1 ester; (e)providing an R1-specific hydrolase, and (f) contacting the 1,3-DAG ofstep (c) with the R1 ester of step (d) and the R1-specific hydrolase ofstep (e) under conditions wherein the R1-specific hydrolase catalyzesesterification of the Sn2 position, thereby producing the structuredlipid. The hydrolase can be an Sn2-specific lipase. The structured lipidcan comprise a cocoa butter alternative (CBA), a synthetic cocoa butter,a natural cocoa butter, 1,3-dipalmitoyl-2-oleoylglycerol (POP),1,3-distearoyl-2-oleoylglycerol (SOS),1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS) or1-oleoyl-2,3-dimyristoylglycerol (OMM).

The invention provides a method for biocatalytic synthesis of astructured lipid comprising the following steps: (a) providing ahydrolase of the invention; (b) providing a composition comprising atriacylglyceride (TAG); (c) contacting the polypeptide of step (a) withthe composition of step (b) under conditions wherein the polypeptidehydrolyzes an acyl residue at the Sn1 or Sn3 position of thetriacylglyceride (TAG), thereby producing a 1,2-DAG or 2,3-DAG; and (d)promoting of acyl migration in the 1,2-DAG or 2,3-DAG of the step (c)under kinetically controlled conditions, thereby producing a 1,3-DAG.The method can further comprise providing an R1 ester and an R1-specificlipase, and contacting the 1,3-DAG of step (d) with the R1 ester and theR1-specific lipase under conditions wherein the R1-specific lipasecatalyzes esterification of the Sn2 position, thereby producing astructured lipid. The lipase can be an Sn1 or an Sn3-specific lipase.The structured lipid can comprise a cocoa butter alternative (CBA), asynthetic cocoa butter, a natural cocoa butter,1,3-dipalmitoyl-2-oleoylglycerol (POP), 1,3-distearoyl-2-oleoylglycerol(SOS), 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS) or1-oleoyl-2,3-dimyristoylglycerol (OMM). In one aspect of the method,step (d) further comprises using ion exchange resins. The kineticallycontrolled conditions can comprise non-equilibrium conditions resultingin production of an end product having greater than a 2:1 ratio of1,3-DAG to 2,3-DAG. The composition of step (b) can comprise afluorogenic fatty acid (FA). The composition of step (b) can comprise anumbelliferyl FA ester. The end product can be enantiomerically pure.

The invention provides a method for preparation of an optical isomer ofa propionic acid from a racemic ester of the propionic acid comprisingthe following steps: (a) providing a hydrolase of the invention, whereinthe hydrolase is stereoselective for an optical isomer of the propionicacid; (b) providing racemic esters; (c) contacting the polypeptide ofstep (a) with the racemic esters of step (b) wherein the polypeptide canselectively catalyze the hydrolysis of the esters of step (b), therebyproducing the optical isomer of the propionic acid. The optical isomerof the propionic acid can comprise an S(+) of 2-(6-methoxy-2-naphthyl)propionic acid and the racemic esters comprises racemic (R,S) esters of2-(6-methoxy-2-naphthyl) propionic acid.

The invention provides a method for stereoselectively hydrolyzingracemic mixtures of esters of 2-substituted acids comprising thefollowing steps: (a) providing a hydrolase of the invention, wherein thehydrolase is stereoselective; (b) providing a composition comprising aracemic mixture of esters of 2-substituted acids; and (c) contacting thepolypeptide of step (a) with the composition of step (b) underconditions wherein the polypeptide of step (b) can selectively hydrolyzethe esters. The hydrolase can be immobilized. The 2-substituted acid cancomprise a 2-aryloxy substituted acid, anR-2-(4-hydroxyphenoxy)propionic acid or a 2-arylpropionic acid. The2-substituted acid can comprise a ketoprofen.

The invention provides a method for oil or fat modification comprisingthe following steps: (a) providing a hydrolase of the invention; (b)providing an oil or fat, and (c) contacting the hydrolase of step (a)with the oil or fat of step (b) under conditions wherein the hydrolasecan modify the oil or fat. The modification can comprise ahydrolase-catalyzed hydrolysis of the fat or oil. The hydrolysis can bea complete or a partial hydrolysis of the fat or oil. The oil cancomprise a glycerol ester of a polyunsaturated fatty acid, or a fish,animal, or vegetable oil. The vegetable oil can comprise an olive,canola, sunflower, palm, soy or lauric oil or rice bran oil.

The invention provides a method for hydrolysis of polyunsaturated fattyacid (PUFA) esters comprising the following steps: (a) providing ahydrolase of the invention; (b) providing composition comprising apolyunsaturated fatty acid ester, and (c) contacting the hydrolase withthe composition of step (b) under conditions wherein the hydrolase canhydrolyze the polyunsaturated fatty acid (PUFA) ester. The inventionprovides a method of selective hydrolysis of polyunsaturated fatty acidsesters over saturated fatty acid esters comprising the following steps:(a) providing a hydrolase of the invention, wherein the hydrolase has alipase activity and selectively hydrolyzes polyunsaturated fatty acid(PUFA) esters; (b) providing a composition comprising a mixture ofpolyunsaturated and saturated esters; and (c) contacting the polypeptideof step (a) with the composition of step (b) under conditions whereinthe polypeptide can selectively catalyze the hydrolysis ofpolyunsaturated fatty acids esters.

The invention provides a method for preparing a food or a feed additivecomprising polyunsaturated fatty acids (PUFA) comprising the followingsteps: (a) providing a hydrolase of the invention, wherein the hydrolaseselectively hydrolyzes polyunsaturated fatty acid (PUFA) esters; (b)providing a composition comprising a PUFA ester; and (c) contacting thepolypeptide of step (a) with the composition of step (b) underconditions wherein the polypeptide can selectively catalyze thehydrolysis of polyunsaturated fatty acid esters thereby producing thePUFA-containing food or feed additive.

The invention provides a method for treatment of latex comprising thefollowing steps: (a) providing a hydrolase of the invention, wherein thepolypeptide has selectivity for a saturated ester over an unsaturatedester, thereby converting the saturated ester to its corresponding acidand alcohol; (b) providing a latex composition comprising saturated andunsaturated esters; (c) contacting the hydrolase of step (a) with thecomposition of step (b) under conditions wherein the polypeptide canselectively hydrolyze saturated esters, thereby treating the latex. Theethyl propionate can be selectively hydrolyzed over ethyl acrylate. Thelatex composition of step (b) can comprise polymers containing acrylic,vinyl and unsaturated acid monomers, alkyl acrylate monomers, methylacrylate, ethyl acrylate, propyl acrylate and butyl acrylate, acrylateacids, acrylic acid, methacrylic acid, crotonic acid, itaconic acid andmixtures thereof. The latex composition can be a hair fixative. Theconditions of step (c) can comprise a pH in the range from about pH 4 topH 8 and a temperature in the range from about 20° to about 50° C.

The invention provides a method for refining a lubricant comprising thefollowing steps: (a) providing a composition comprising a hydrolase ofthe invention; (b) providing a lubricant; and (c) treating the lubricantwith the hydrolase under conditions wherein the hydrolase can selectivehydrolyze oils in the lubricant, thereby refining it. The lubricant canbe a hydraulic oil.

The invention provides a method of treating a fabric comprising thefollowing steps: (a) providing a composition comprising a hydrolase ofthe invention, wherein the hydrolase can selectively hydrolyzecarboxylic esters; (b) providing a fabric; and (c) treating the fabricwith the hydrolase under condition wherein the hydrolase can selectivelyhydrolyze carboxylic esters thereby treating the fabric. The treatmentof the fabric can comprise improvement of the hand and drape of thefinal fabric, dyeing, obtaining flame retardancy, obtaining waterrepellency, obtaining optical brightness, or obtaining resin finishing.The fabric can comprise cotton, viscose, rayon, lyocell, flax, linen,ramie, all blends thereof, or blends thereof with polyesters, wool,polyamides acrylics or polyacrylics. The invention provides a fabric,yarn or fiber comprising a hydrolase of the invention, which can beadsorbed, absorbed or immobilized on the surface of the fabric, yarn orfiber.

The invention provides a method for removing or decreasing the amount ofa food or oil stain comprising contacting a hydrolase of the inventionwith the food or oil stain under conditions wherein the hydrolase canhydrolyze oil or fat in the stain. The hydrolase can have an enhancedstability to denaturation by surfactants and to heat deactivation. Thehydrolase can have a detergent or a laundry solution.

The invention provides a dietary composition comprising a hydrolase ofthe invention. The dietary composition can further comprise anutritional base comprising a fat. The hydrolase can be activated by abile salt. The dietary composition can further comprising a cow'smilk-based infant formula. The hydrolase can hydrolyze long chain fattyacids. The invention provides a method of reducing fat content in milkor vegetable-based dietary compositions comprising the following steps:(a) providing a composition comprising a hydrolase of the invention; (b)providing a composition comprising a milk or a vegetable oil, and (c)treating the composition of step (b) with the hydrolase under conditionswherein the hydrolase can hydrolyze the oil or fat in the composition,thereby reducing its fat content. The invention provides a dietarycomposition for a human or non-ruminant animals comprising a nutritionalbase, wherein the base comprises a fat and no or little hydrolase, andan effective amount of a hydrolase to increase fat absorption and growthof human or non-ruminant animal.

The invention provides a method of catalyzing an interesterificationreaction to produce new triglycerides comprising the following steps:(a) providing a composition comprising a hydrolase of the invention,wherein the hydrolase can catalyze an interesterification reaction; (b)providing a mixture of triglycerides and free fatty acids; (c) treatingthe composition of step (b) with the hydrolase under conditions whereinthe hydrolase can catalyze exchange of free fatty acids with the acylgroups of triglycerides, thereby producing new triglycerides enriched inthe added fatty acids. The hydrolase can be an Sn1,3-specific lipase.The invention provides a transesterification method for preparing amargarine oil having a low trans-acid and a low intermediate chain fattyacid content, comprising the following steps: (a) providing atransesterification reaction mixture comprising a stearic acid sourcematerial selected from the group consisting of stearic acid, stearicacid monoesters of low molecular weight monohydric alcohols and mixturesthereof, (b) providing a liquid vegetable oil; (c) providing a hydrolaseof the invention, wherein the polypeptide comprises a 1,3-specificlipase activity; (d) transesterifying the stearic acid source materialand the vegetable oil triglyceride, to substantially equilibrate theester groups in the 1-, 3-positions of the glyceride component withnon-glyceride fatty acid components of the reaction mixture, (e)separating transesterified free fatty acid components from glyceridecomponents of the transesterification mixture to provide atransesterified margarine oil product and a fatty acid mixturecomprising fatty acids, fatty acid monoesters or mixtures thereofreleased from the vegetable oil, and (f) hydrogenating the fatty acidmixture.

The invention provides a method for making a composition comprising1-palmitoyl-3-stearoyl-2-monoleine (POSt) and 1,3-distearoyl-2-monoleine(StOSt) comprising providing a lipase, wherein the lipase is capable of1,3-specific lipase-catalyzed interesterification of1,3-dipalmitoyl-2-monoleine (POP) with stearic acid or tristearin, tomake a product enriched in the 1-palmitoyl-3-stearoyl-2-monoleine (POSt)or 1,3-distearoyl-2-monoleine (StOSt).

The invention provides a method for ameliorating or preventinglipopolysaccharide (LPS)-mediated toxicity comprising administering to apatient a pharmaceutical composition comprising a polypeptide of theinvention. The invention provides a method for detoxifying an endotoxincomprising contacting the endotoxin with a polypeptide of the invention.The invention provides a method for deacylating a 2′ or a 3′ fatty acidchain from a lipid A comprising contacting the lipid A with apolypeptide of the invention.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

DESCRIPTION OF DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a block diagram of a computer system.

FIG. 2 is a flow diagram illustrating one aspect of a process forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 3 is a flow diagram illustrating one aspect of a process in acomputer for determining whether two sequences are homologous.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence.

FIG. 5 illustrates an exemplary method of the invention to test forlipase activity, a colorimetric lipase assay, as described in Example 1,below.

FIG. 6 illustrates an exemplary method of the invention using an Sn2regia-specific lipase in the synthesis of structured lipids.

FIG. 7 illustrates an exemplary method of the invention, a “ForcedMigration Methodology” for the structured synthesis of lipids, asdescribed in detail in Example 2, below.

FIG. 8 illustrates an exemplary method comprising use of lipases of theinvention to synthesize cocoa butter alternatives, as described indetail below.

FIG. 9A illustrates exemplary methods of the invention comprisingsynthesizing PUPA-containing sTAGs.

FIG. 9B illustrates exemplary methods of the invention comprisingsynthesizing 2-PUFA sMAGs and purified PUFAs.

FIG. 10 illustrates an exemplary method comprising a coupled enzymeassay, as discussed in detail, below.

FIG. 11 illustrates an exemplary growth-kill assay, as discussed inExample 6, below.

FIG. 12 illustrates data of various esterification reactions in thesynthesis of 1,3-DCy, as discussed in Example 7, below.

FIG. 13 summarizes data showing the effect of substrate ratio onesterification between glycerol and caprylic acid, as discussed inExample 7, below.

FIG. 14 summarizes data of various synthesis of 1,3-dilaurin, asdiscussed in Example 7, below.

FIG. 15 summarizes the effect of substrate ration on esterification ofglycerol and lauric acid inn-hexane, as discussed in Example 7, below.

FIG. 16 summarizes the synthesis of 1,3-dipalmitin, as discussed inExample 7, below.

FIG. 17 summarizes data for the esterification of glycerol and palmitic(C16:0) or stearic (C18:0) acid, as discussed in Example 7, below.

FIG. 18 shows data from alcoholysis reaction, as discussed in Example 7,below.

FIG. 19 illustrates data from the hydrolysis of trilaurin, as discussedin Example 7, below.

FIG. 20 shows the effect of trilaurin:water ratio on hydrolysis oftrilaurin, as discussed in Example 7, below.

FIG. 21 summarizes data showing the effect of organic solvents onhydrolysis of trilaurin, as discussed in Example 7, below.

FIG. 22 illustrates data from the alcoholysis and hydrolysis of coconutoil in organic solvent, as discussed in Example 7, below.

FIG. 23 shows the effect of oleic acid on acyl migration of1,2-dipalmitin in n-hexane at room temperature, as discussed in Example7, below.

FIG. 24 shows the effect of the amount of anion exchanger on acylmigration of 1,2-dipalmitin in n-hexane, as discussed in Example 7,below.

FIG. 25 shows data from the esterification of 1,3-dicaprylin and oleicacid vinyl ester in n-hexane by an immobilized lipase from a Pseudomonassp. (Amano PS-D, Amano Enzyme USA, Elgin, Ill.), as discussed in Example7, below.

FIG. 26 shows data from the esterification of 1,3-DG and oleic acid oroleic acid vinyl ester in n-hexane by an immobilized lipase from aPseudomonas sp. (Amano PS-D, Amano Enzyme USA, Elgin, Ill.), asdiscussed in Example 7, below.

FIG. 27 illustrates an exemplary forced migration reaction of theinvention, as discussed below.

FIG. 28 illustrates an exemplary synthesis of a triglyceride mixturecomposed of POS (Palmitic-Oleic-Stearic), POP (Palmitic-Oleic-Palmitic)and SOS (Stearic-Oleic-Stearic) from glycerol, as discussed below.

FIG. 29 illustrates an exemplary synthesis where stearate and palmitateare mixed together to generate mixtures of DAGs which are subsequentlyacylated with oleate to give components of cocoa butter equivalents, asdiscussed below.

FIG. 30 is a chart describing selected characteristics of exemplarynucleic acids and polypeptides of the invention, as described in furtherdetail, below.

FIG. 31 schematically illustrates data from a two enzyme system of theinvention, as described in Example 10, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

In one aspect, the invention provides hydrolases, polynucleotidesencoding them, and methods of making and using these polynucleotides andpolypeptides. In one aspect, the invention is directed to polypeptides,e.g., enzymes, having a hydrolase activity, e.g., an esterase, acylase,lipase, phospholipase or protease activity, including thermostable andthermotolerant hydrolase activity, and polynucleotides encoding theseenzymes, and making and using these polynucleotides and polypeptides.The hydrolase activities of the polypeptides and peptides of theinvention include esterase activity, lipase activity (hydrolysis oflipids), acidolysis reactions (to replace an esterified fatty acid witha free fatty acid), transesterification reactions (exchange of fattyacids between triglycerides), ester synthesis, ester interchangereactions, phospholipase activity (e.g., phospholipase A, B, C and Dactivity, patatin activity, lipid acyl hydrolase (LAH) activity) andprotease activity (hydrolysis of peptide bonds). The polypeptides of theinvention can be used in a variety of pharmaceutical, agricultural andindustrial contexts, including the manufacture of cosmetics andnutraceuticals. In another aspect, the polypeptides of the invention areused to synthesize enantiomerically pure chiral products. Thepolypeptides of the invention can be used in a variety ofpharmaceutical, agricultural and industrial contexts, including themanufacture of cosmetics and nutraceuticals.

Enzymes of the invention can be highly selective catalysts. They canhave the ability to catalyze reactions with stereo-, regio-, andchemo-selectivities not possible in conventional synthetic chemistry.Enzymes of the invention can be versatile. In various aspects, they canfunction in organic solvents, operate at extreme pHs (for example, highpHs and low pHs) extreme temperatures (for example, high temperaturesand low temperatures), extreme salinity levels (for example, highsalinity and low salinity), and catalyze reactions with compounds thatare structurally unrelated to their natural, physiological substrates.

Hydrolases of the Invention Having Lipase Activity

In one aspect, the polypeptides of the invention have lipase activityand can be used as lipases, e.g., in the biocatalytic synthesis ofstructured lipids (lipids that contain a defined set of fatty acidsdistributed in a defined manner on the glycerol backbone), includingcocoa butter alternatives, poly-unsaturated fatty acids (PUFAs),1,3-diacyl glycerides (DAGs), 2-monoglycerides (MAGs) andtriacylglycerides (TAGs), such as 1,3-dipalmitoyl-2-oleoylglycerol(POP), 1,3-distearoyl-2-oleoylglycerol (SOS),1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS) or1-oleoyl-2,3-dimyristoylglycerol (OMM), long chain polyunsaturated fattyacids such as arachidonic acid, docosahexaenoic acid (DHA) andeicosapentaenoic acid (EPA).

In one aspect, the invention provides an exemplary synthesis (usinglipases of the invention) of a triglyceride mixture composed of POS(Palmitic-Oleic-Stearic), POP (Palmitic-Oleic-Palmitic) and SOS(Stearic-Oleic-Stearic) from glycerol, as illustrated in FIG. 28. Thisexemplary synthesis uses free fatty acids versus fatty acid esters. Inone aspect, this reaction can be performed in one pot with sequentialaddition of fatty acids using crude glycerol and free fatty acids andfatty acid esters. In one aspect, stearate and palmitate are mixedtogether to generate mixtures of DAGs. In one aspect, thediacylglycerides are subsequently acylated with oleate to givecomponents of cocoa butter equivalents, as illustrated in FIG. 29. Inalternative aspects, the proportions of POS, POP and SOS can be variedaccording to: stearate to palmitate ratio; selectivity of enzyme forpalmitate versus stearate; or enzyme enantioselectivity (could alterlevels of POS/SOP). One-pot synthesis of cocoa butter equivalents orother cocoa butter alternatives is possible using this aspect of theinvention.

In one aspect, lipases that exhibit regioselectivity and/orchemoselectivity are used in the structure synthesis of lipids or in theprocessing of lipids. Thus, the methods of the invention use lipaseswith defined regio-specificity or defined chemoselectivity (e.g., afatty acid specificity) in a biocatalytic synthetic reaction. Forexample, the methods of the invention can use lipases with SN1, SN2and/or SN3 regio-specificity, or combinations thereof. In one aspect,the methods of the invention use lipases that exhibit regioselectivityfor the 2-position of a triacylglyceride (TAG). This SN2regioselectivity can be used in the synthesis of a variety of structuredlipids, e.g., triacylglycerides (TAGs), including 1,3-DAGs andcomponents of cocoa butter, as illustrated in FIG. 6.

The methods and compositions (lipases) of the invention can be used inthe biocatalytic synthesis of structured lipids, and the production ofnutraceuticals (e.g., polyunsaturated fatty acids and oils), variousfoods and food additives (e.g., emulsifiers, fat replacers, margarinesand spreads), cosmetics (e.g., emulsifiers, creams), pharmaceuticals anddrug delivery agents (e.g., liposomes, tablets, formulations), andanimal feed additives (e.g., polyunsaturated fatty acids, such aslinoleic acids) comprising lipids made by the structured synthesismethods of the invention or processed by the methods of the invention

In one aspect, lipases of the invention can act on fluorogenic fattyacid (FA) esters, e.g., umbelliferyl FA esters. In one aspect, profilesof FA specificities of lipases made or modified by the methods of theinvention can be obtained by measuring their relative activities on aseries of umbelliferyl FA esters, such as palmitate, stearate, oleate,laurate, PUFA, butyrate. The following table 1 indicates activity forsome exemplary lipases of the invention. The activity was tested onbutyrate and oleate.

TABLE 1 Activity for exemplary lipases with umbelliferyl FA estersSubstrate Specific Substrate Concen- SEQ ID Specific Activity Concen-tration NO: Activity Units Substrate tration Units 69, 70 2713.1 U/mgMuf- 1 mM Butyrate 67, 68 51.1 U/mg Muf-Oleate 1 mM 67, 68 5451.2 U/mgMuf- 1 mM Butyrate 99, 100 2441 U/mg Muf- 1 mM Butyrate 99, 100 13 U/mgMuf-Oleate 1 mM 15, 16 1847.7 U/mg Muf- 1 mM Butyrate 15, 16 43.2 U/mgMuf-Oleate 1 mM 39, 40 11560.8 U/mg Muf- 1 mM Butyrate 39, 40 1601.2U/mg Muf-Oleate 1 mM 75, 76 25842.6 U/mg Muf- 1 mM Butyrate 75, 76 3.7U/mg Muf-Oleate 1 mM 33, 34 28769.7 U/mg Muf- 1 mM Butyrate 33, 34 4.6U/mg Muf-Oleate 1 mM 25, 26 8.9 U/mg Muf-Oleate 1 mM 25, 26 49382.7 U/mgMuf- 1 mM Butyrate 3, 4 1193.1 U/mg Muf- 1 mM Butyrate 3, 4 97 U/mgMuf-Oleate 1 mM 113, 114 0.12 U/mg Muf-Oleate 1 mM 113, 114 95.5 U/mgMuf- 1 mM Butyrate

The methods and compositions (lipases) of the invention can be used tosynthesize enantiomerically pure chiral products. In one aspect, themethods and compositions (lipases) of the invention can be used toprepare a D-amino acid and corresponding esters from a racemic mix. Forexample, D-aspartic acid can be prepared from racemic aspartic acid. Inone aspect, optically active D-homophenylalanine and/or its esters areprepared. The enantioselectively synthesized D-homophenylalanine can bestarting material for many drugs, such as Enalapril, Lisinopril, andQuinapril, used in the treatment of hypertension and congestive heartfailure. The D-aspartic acid and its derivatives made by the methods andcompositions of the invention can be used in pharmaceuticals, e.g., forthe inhibition of arginiosuccinate synthetase to prevent or treat sepsisor cytokine-induced systemic hypotension or as immunosuppressive agents.The D-aspartic acid and its derivatives made by the methods andcompositions of the invention can be used as taste modifyingcompositions for foods, e.g., as sweeteners (e.g., ALITAME™). Forexample, the methods and compositions (lipases) of the invention can beused to synthesize an optical isomer S(+) of 2-(6-methoxy-2-naphthyl)propionic acid from a racemic (R,S) ester of 2-(6-methoxy-2-naphthyl)propionic acid (see, e.g., U.S. Pat. No. 5,229,280).

In one aspect, the methods and compositions (lipases) of the inventioncan be used to for stereoselectively hydrolyzing racemic mixtures ofesters of 2-substituted acids, e.g., 2-aryloxy substituted acids, suchas R-2-(4-hydroxyphenoxy)propionic acid, 2-arylpropionic acid,ketoprofen to synthesize enantiomerically pure chiral products. See,e.g., U.S. Pat. No. 5,108,916.

In one aspect, the lipase of the invention for these reactions isimmobilized, e.g., as described below. In alternative aspects, themethods of the invention do not require an organic solvent, can proceedwith relatively fast reaction rates; and do not require a protectivegroup for the amino acid. See, e.g., U.S. Pat. Nos. 5,552,317;5,834,259.

The methods and compositions (lipases) of the invention can be used tohydrolyze oils, such as fish, animal and vegetable oils, and lipids,such as poly-unsaturated fatty acids. In one aspect, the polypeptides ofthe invention are used process fatty acids (such as poly-unsaturatedfatty acids), e.g., fish oil fatty acids, for use in or as a feedadditive. Addition of poly-unsaturated fatty acids PUFAs to feed fordairy cattle has been demonstrated to result in improved fertility andmilk yields. Fish oil contains a high level of PUFAs (see Table 2,below) and therefore is a potentially inexpensive source for PUFAs as astarting material for the methods of the invention. The biocatalyticmethods of the invention can process fish oil under mild conditions,thus avoiding harsh conditions utilized in some processes. Harshconditions may promote unwanted isomerization, polymerization andoxidation of the PUFAs. In one aspect, the methods of the inventioncomprise lipase-catalyzed total hydrolysis of fish-oil or selectivehydrolysis of PUFAs from fish oil to provide a mild alternative thatwould leave the high-value PUFAs intact. In one aspect, the methodsfurther comprise hydrolysis of lipids by chemical or physical splittingof the fat.

TABLE 2 Fatty acid composition of a variety of fats and oils. Fatty acidcontent of fats (%): 14:0 16:0 16:1 18:0 18:1 18:2 18:3 18:4 20:x 22:xTallow 3.7 24.9 4.2 18.9 36 3.1 Lard 23.8 2.7 13.5 41.2 10.2 1.0 CanolaOil 4.0 1.8 56.1 20.3 9.3 2.4 1.0 Soybean Oil 10.3 3.8 22.8 51 6.8 0.2Palm Oil 48.6 4.1 36.6 9.1 0.3 0.1 Corn Oil 10.9 1.8 24.2 58 0.7 FishOil 7.2 16.7 11.1 3.2 10.2 1.4 2.4 3.5 16.4 16.1

In one aspect, the lipases and methods of the invention are used for thetotal hydrolysis of fish oil. Lipases can be screened for their abilityto catalyze the total hydrolysis of fish oil under different conditionsusing, e.g., a method comprising a coupled enzyme assay, as illustratedin FIG. 10, to detect the release of glycerol from lipids. This assayhas been validated in the presence of lipid emulsions and retainssensitivity under these conditions. In alternative aspects, a single ormultiple lipases are used to catalyze the total splitting of the fishoil. Several lipases of the invention may need to be used, owing to thepresence of the PUFAs. In one aspect, a PUFA-specific lipase of theinvention is combined with a general lipase to achieve the desiredeffect.

The methods and compositions (lipases) of the invention can be used tocatalyze the partial or total hydrolysis of other oils, e.g. olive oils,that do not contain PUFAs.

The methods and compositions (lipases) of the invention can be used tocatalyze the hydrolysis of PUFA glycerol esters. These methods can beused to make feed additives. In one aspect, lipases of the inventioncatalyze the release of PUFAs from simple esters and fish oil. Standardassays and analytical methods can be utilized.

The methods and compositions (lipases) of the invention can be used toselectively hydrolyze saturated esters over unsaturated esters intoacids or alcohols. The methods and compositions (lipases) of theinvention can be used to treat latexes for a variety of purposes, e.g.,to treat latexes used in hair fixative compositions to remove unpleasantodors. The methods and compositions (lipases) of the invention can beused in the treatment of a lipase deficiency in an animal, e.g., amammal, such as a human. The methods and compositions (lipases) of theinvention can be used to prepare lubricants, such as hydraulic oils. Themethods and compositions (lipases) of the invention can be used inmaking and using detergents. The methods and compositions (lipases) ofthe invention can be used in processes for the chemical finishing offabrics, fibers or yarns. In one aspect, the methods and compositions(lipases) of the invention can be used for obtaining flame retardancy ina fabric using, e.g., a halogen-substituted carboxylic acid or an esterthereof, i.e. a fluorinated, chlorinated or bromated carboxylic acid oran ester thereof. In one aspect, the invention provides methods ofgenerating lipases from environmental libraries.

Hydrolases of the Invention Having Esterase or Acylase Activity

In one aspect, the hydrolase activity of the invention comprises anacylase or an esterase activity. In one aspect, the hydrolysis activitycomprises hydrolyzing a lactone ring or acylating an acyl lactone or adiol lactone. In one aspect, the hydrolysis activity comprises anesterase activity. In one aspect, the esterase activity compriseshydrolysis of ester groups to organic acids and alcohols. In one aspect,the esterase activity comprises feruloyl esterase activity. In oneaspect, the esterase activity comprises a lipase activity. Inalternative aspects, the esterase activities of the enzymes of theinvention include lipase activity (in the hydrolysis of lipids),acidolysis reactions (to replace an esterified fatty acid with a freefatty acid), transesterification reactions (exchange of fatty acidsbetween triglycerides), ester synthesis and ester interchange reactions.The enzymes of the invention can also be utilized in organic synthesisreactions in the manufacture of medicaments, pesticides or intermediatesthereof.

In one aspect, the polypeptides of the invention have esterase oracylase activity and can be used, e.g., to hydrolyze a lactone ring oracylate an acyl lactone or a diol lactone. In one aspect, the hydrolysisactivity of a polypeptide of the invention comprises an esteraseactivity. In one aspect, the esterase activity comprises hydrolysis ofester groups to organic acids and alcohols. In one aspect, the esteraseactivity comprises feruloyl esterase activity. In one aspect, theesterase activity comprises a lipase activity. In alternative aspects,the esterase activities of the enzymes of the invention include lipaseactivity (in the hydrolysis of lipids), acidolysis reactions (to replacean esterified fatty acid with a free fatty acid), transesterificationreactions (exchange of fatty acids between triglycerides), estersynthesis and ester interchange reactions. The enzymes of the inventioncan also be utilized in organic synthesis reactions in the manufactureof medicaments, pesticides or intermediates thereof. The esteraseactivities of the polypeptides and peptides of the invention includelipase activity (in the hydrolysis of lipids), acidolysis reactions (toreplace an esterified fatty acid with a free fatty acid),transesterification reactions (exchange of fatty acids betweentriglycerides), ester synthesis and ester interchange reactions. Thepolypeptides and peptides of the invention can also be utilized inorganic synthesis reactions in the manufacture of medicaments,pesticides or intermediates thereof.

Hydrolases of the Invention Having Protease Activity

In one aspect, the invention provides polypeptides having a proteaseactivity, polynucleotides encoding the polypeptides, and methods formaking and using these polynucleotides and polypeptides. In one aspect,the proteases of the invention are used to catalyze the hydrolysis ofpeptide bonds. The proteases of the invention can be used to make and/orprocess foods or feeds, textiles, detergents and the like. The proteasesof the invention can be used in pharmaceutical compositions and dietaryaids.

The protease preparations of the invention (including those for treatingor processing feeds or foods, treating fibers and textiles, wastetreatments, plant treatments, and the like) can further comprise one ormore enzymes, for example, pectate lyases, cellulases(endo-beta-1,4-glucanases), beta-glucanases(endo-beta-1,3(4)-glucanases), lipases, cutinases, peroxidases,laccases, amylases, glucoamylases, pectinases, reductases, oxidases,phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,mannanases, xyloglucanases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, polygalacturonases,rhamnogalacturonases, galactanases, pectin lyases, pectinmethylesterases, cellobiohydrolases, transglutaminases; or mixturesthereof.

A polypeptide can be routinely assayed for protease activity (e.g.,tested to see if the protein is within the scope of the invention) byany method, e.g., protease activity can be assayed by the hydrolysis ofcasein in zymograms, the release of fluorescence from gelatin, or therelease of p-nitroanalide from various small peptide substrates (theseand other exemplary protease assays are set forth in the Examples,below).

Hydrolases of the Invention Having Phospholipase Activity

In one aspect, the invention provides polypeptides having aphospholipase activity. The phospholipases of the invention can havephospholipase A, B, C, D, a lipid acyl hydrolase (LAH), or patatinenzyme activity. The phospholipases of the invention can efficientlycleave glycerolphosphate ester linkage in oils, such as vegetable oils,e.g., oilseed phospholipids, to generate a water extractablephosphorylated base and a diglyceride. In alternative aspects, thephospholipases of the invention can cleave glycerolphosphate esterlinkages in phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine and sphingomyelin.

In one aspect, the phospholipases of the invention are used in variousvegetable oil processing steps, such as in vegetable oil extraction,particularly, in the removal of “phospholipid gums” in a process called“oil degumming,” as described herein. The production of vegetable oilsfrom various sources, such as rice bran, soybeans, rapeseed, peanut,sesame, sunflower and corn. The phospholipase enzymes of the inventioncan be used in place of PLA, e.g., phospholipase A2, in any vegetableoil processing step. A phospholipase of the invention (e.g.,phospholipase A, B, C, D, patatin enzymes) can be used for enzymaticdegumming of vegetable oils because the phosphate moiety is soluble inwater and easy to remove. The diglyceride product will remain in the oiland therefore will reduce losses. The PLCs of the invention can be usedin addition to or in place of PLA1s and PLA2s in commercial oildegumming, such as in the ENZYMAX® process, where phospholipids arehydrolyzed by PLA1 and PLA2.

In alternative aspects, enzymes of the invention havephosphatidylinositol-specific phospholipase C (PI-PLC) activity,phosphatidylcholine-specific phospholipase C activity, phosphatidic acidphosphatase activity, phospholipase A activity and/or patatin-relatedphospholipase activity. These enzymes can be used alone or incombination each other or with other enzymes of the invention, or otherenzymes. In one aspect, the invention provides methods wherein theseenzymes (including phosphatidylinositol-specific phospholipase C,phosphatidylcholine-specific phospholipase C, phosphatidic acidphosphatase, phospholipase A and/or patatin-related phospholipases ofthe invention) are used alone or in combination in the degumming ofoils, e.g., rice bran oil, vegetable oils, e.g., high phosphorous oils,such as soybean, corn, canola and sunflower oils.

These enzymes and processes of the invention can be used to achieve amore complete degumming of high phosphorous oils, in particular, ricebran, soybean, corn, canola, and sunflower oils. Upon cleavage byPI-PLC, phosphatidylinositol is converted to diacylglycerol andphosphoinositol. The diacylglycerol partitions to the aqueous phase(improving oil yield) and the phosphoinositol partitions to the aqueousphase where it is removed as a component of the heavy phase duringcentrifugation. An enzyme of the invention, e.g., a PI-PLC of theinvention, can be incorporated into either a chemical or physical oilrefining process.

In one aspect, hydrolases, e.g., PLC phospholipases, of the inventionutilize a variety of phospholipid substrates includingphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, and phosphatidic acid. In addition, these enzymescan have varying degrees of activity on the lysophospholipid forms ofthese phospholipids. In various aspects, PLC enzymes of the inventionmay show a preference for phosphatidylcholine andphosphatidylethanolamine as substrates.

In one aspect, hydrolases, e.g., phosphatidylinositol PLCphospholipases, of the invention utilize a variety of phospholipidsubstrates including phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Inaddition, these enzymes can have varying degrees of activity on thelysophospholipid forms of these phospholipids. In various aspects,phosphatidylinositol PLC enzymes of the invention may show a preferencefor phosphatidylinositol as a substrate.

In one aspect, hydrolases, e.g., patatin enzymes, of the inventionutilize a variety of phospholipid substrates includingphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, and phosphatidic acid. In addition, these enzymescan have varying degrees of activity on the lysophospholipid forms ofthese phospholipids. In various aspects, patatins of the invention arebased on a conservation of amino acid sequence similarity. In variousaspects, these enzymes display a diverse set of biochemical propertiesand may perform reactions characteristic of PLA1, PLA2, PLC, or PLDenzyme classes.

In one aspect, hydrolases, e.g., PLD phospholipases, of the inventionutilize a variety of phospholipid substrates includingphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, and phosphatidic acid. In addition, these enzymescan have varying degrees of activity on the lysophospholipid forms ofthese phospholipids. In one aspect, these enzymes are useful forcarrying out transesterification reactions to produce structuredphospholipids.

DEFINITIONS

As used herein, the term “hydrolase” encompasses polypeptides (e.g.,antibodies, enzymes) and peptides (e.g., “active sites”) having anyhydrolase activity, i.e., the polypeptides of the invention can have anyhydrolase activity, including lipase, esterase, phospholipase and/orprotease activity.

The term “lipase” includes all polypeptides having any lipase activity,including lipid synthesis or lipid hydrolysis activity, i.e., thepolypeptides of the invention can have any lipase activity. Lipases ofthe invention include enzymes active in the bioconversion of lipidsthrough catalysis of hydrolysis, alcoholysis, acidolysis, esterificationand aminolysis reactions. In one aspect, lipases of the invention canhydrolyze lipid emulsions. In one aspect, enzymes of the invention canact preferentially on sn-1 and/or sn-3 bonds of triglycerides to releasefatty acids from the glycerol backbone. For example, lipase activity ofthe polypeptides of the invention include synthesis of cocoa butter,poly-unsaturated fatty acids (PUFAs), 1,3-diacyl glycerides (DAGs),2-monoglycerides (MAGs) and triacylglycerides (TAGs). The term alsoincludes lipases capable of isomerizing bonds at high temperatures, lowtemperatures, alkaline pHs and at acidic pHs.

The term “phospholipase” encompasses enzymes having any phospholipaseactivity, i.e., the polypeptides of the invention can have anyphospholipase activity. For example, a phospholipase activity of theinvention can comprise cleaving a glycerolphosphate ester linkage(catalyzing hydrolysis of a glycerolphosphate ester linkage), e.g., inan oil, such as a vegetable oil. A phospholipase activity of theinvention can generate a water extractable phosphorylated base and adiglyceride. A phospholipase activity of the invention also includeshydrolysis of glycerolphosphate ester linkages at high temperatures, lowtemperatures, alkaline pHs and at acidic pHs. The term “a phospholipaseactivity” also includes cleaving a glycerolphosphate ester to generate awater extractable phosphorylated base and a diglyceride. The term “aphospholipase activity” also includes cutting ester bonds of glycerinand phosphoric acid in phospholipids. The term “a phospholipaseactivity” also includes other activities, such as the ability to bind toa substrate, such as an oil, e.g. a vegetable oil, substrate alsoincluding plant and animal phosphatidylcholines,phosphatidyl-ethanolamines, phosphatidylserines and sphingomyelins. Thephospholipase activity can comprise a phospholipase C (PLC) activity, aphospholipase A (PLA) activity, such as a phospholipase A1 orphospholipase A2 activity, a phospholipase B (PLB) activity, such as aphospholipase B1 or phospholipase B2 activity, a phospholipase D (PLD)activity, such as a phospholipase D1 or a phospholipase D2 activity. Thephospholipase activity can comprise hydrolysis of a glycoprotein, e.g.,as a glycoprotein found in a potato tuber or any plant of the genusSolanum, e.g., Solanum tuberosum. The phospholipase activity cancomprise a patatin enzymatic activity, such as a patatin esteraseactivity (see, e.g., Jimenez (2002) Biotechnol. Prog. 18:635-640). Thephospholipase activity can comprise a lipid acyl hydrolase (LAH)activity.

The term “protease” includes all polypeptides having a proteaseactivity, including a peptidase and/or a proteinase activity; i.e., thepolypeptides of the invention can have any protease activity. A proteaseactivity of the invention can comprise catalysis of the hydrolysis ofpeptide bonds. The proteases of the invention can catalyze peptidehydrolysis reactions in both directions. The direction of the reactioncan be determined, e.g., by manipulating substrate and/or productconcentrations, temperature, selection of protease and the like. Theprotease activity can comprise an endoprotease activity and/or anexoprotease activity. The protease activity can comprise a proteaseactivity, e.g., a carboxypeptidase activity, a dipeptidylpeptidase or anaminopeptidase activity, a serine protease activity, a metalloproteinaseactivity, a cysteine protease activity and/or an aspartic proteaseactivity. In one aspect, protease activity can comprise activity thesame or similar to a chymotrypsin, a trypsin, an elastase, a kallikreinand/or a subtilisin activity.

The term esterase includes all polypeptides having an esterase activity,i.e., the polypeptides of the invention can have any esterase activity.For example, the invention provides polypeptides capable of hydrolyzingester groups to organic acids and alcohols. The term “esterase” alsoencompasses polypeptides having lipase activity (in the hydrolysis oflipids), acidolysis reactions (to replace an esterified fatty acid witha free fatty acid), trans-esterification reactions (exchange of fattyacids between triglycerides), ester synthesis and ester interchangereactions. In one aspect, the hydrolases of the invention can hydrolyzea lactone ring or acylate an acyl lactone or a diol lactone. Thepolypeptides of the invention can be enantiospecific, e.g., as when usedin chemoenzymatic reactions in the synthesis of medicaments andinsecticides. The polynucleotides of the invention encode polypeptideshaving esterase activity.

A hydrolase variant (e.g., “lipase variant”, “esterase variant”,“protease variant” “phospholipase variant”) can have an amino acidsequence which is derived from the amino acid sequence of a “precursor”.The precursor can include naturally-occurring hydrolase and/or arecombinant hydrolase. The amino acid sequence of the hydrolase variantis “derived” from the precursor hydrolase amino acid sequence by thesubstitution, deletion or insertion of one or more amino acids of theprecursor amino acid sequence. Such modification is of the “precursorDNA sequence” which encodes the amino acid sequence of the precursorlipase rather than manipulation of the precursor hydrolase enzyme perse. Suitable methods for such manipulation of the precursor DNA sequenceinclude methods disclosed herein, as well as methods known to thoseskilled in the art.

The term “antibody” includes a peptide or polypeptide derived from,modeled after or substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, capable of specificallybinding an antigen or epitope, see, e.g. Fundamental Immunology, ThirdEdition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J.Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys.Methods 25:85-97. The term antibody includes antigen-binding portions,i.e., “antigen binding sites,” (e.g., fragments, subsequences,complementarity determining regions (CDRs)) that retain capacity to bindantigen, including (i) a Fab fragment, a monovalent fragment consistingof the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso included by reference in the term “antibody.” Thus, the inventionprovides antibodies, including antigen binding sites and single chainantibodies that specifically bind to a hydrolase of the invention. Inpracticing the methods of the invention, polypeptides having a hydrolaseactivity can also be used.

The terms “array” or “microarray” or “biochip” or “chip” as used hereinis a plurality of target elements, each target element comprising adefined amount of one or more polypeptides (including antibodies) ornucleic acids immobilized onto a defined area of a substrate surface, asdiscussed in further detail, below.

As used herein, the terms “computer,” “computer program” and “processor”are used in their broadest general contexts and incorporate all suchdevices, as described in detail, below.

A “coding sequence of” or a “sequence encodes” a particular polypeptideor protein, is a nucleic acid sequence which is transcribed andtranslated into a polypeptide or protein when placed under the controlof appropriate regulatory sequences.

The term “expression cassette” as used herein refers to a nucleotidesequence which is capable of affecting expression of a structural gene(i.e., a protein coding sequence, such as a hydrolase of the invention)in a host compatible with such sequences. Expression cassettes includeat least a promoter operably linked with the polypeptide codingsequence; and, optionally, with other sequences, e.g., transcriptiontermination signals. Additional factors necessary or helpful ineffecting expression may also be used, e.g., enhancers. “Operablylinked” as used herein refers to linkage of a promoter upstream from aDNA sequence such that the promoter mediates transcription of the DNAsequence. Thus, expression cassettes also include plasmids, expressionvectors, recombinant viruses, any form of recombinant “naked DNA”vector, and the like. A “vector” comprises a nucleic acid which caninfect, transfect, transiently or permanently transduce a cell. It willbe recognized that a vector can be a naked nucleic acid, or a nucleicacid complexed with protein or lipid. The vector optionally comprisesviral or bacterial nucleic acids and/or proteins, and/or membranes(e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include,but are not limited to replicons (e.g., RNA replicons, bacteriophages)to which fragments of DNA may be attached and become replicated. Vectorsthus include, but are not limited to RNA, autonomous self-replicatingcircular or linear DNA or RNA (e.g., plasmids, viruses, and the like,see, e.g., U.S. Pat. No. 5,217,879), and includes both the expressionand non-expression plasmids. Where a recombinant microorganism or cellculture is described as hosting an “expression vector” this includesboth extra-chromosomal circular and linear DNA and DNA that has beenincorporated into the host chromosome(s). Where a vector is beingmaintained by a host cell, the vector may either be stably replicated bythe cells during mitosis as an autonomous structure, or is incorporatedwithin the host's genome.

The term “gene” can include a nucleic acid sequence comprising a segmentof DNA involved in producing a transcription product (e.g., a message),which in turn is translated to produce a polypeptide chain, or regulatesgene transcription, reproduction or stability. Genes can include, interalia, regions preceding and following the coding region, such as leaderand trailer, promoters and enhancers, as well as, where applicable,intervening sequences (introns) between individual coding segments(exons).

The phrases “nucleic acid” or “nucleic acid sequence” can include anoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA (e.g., mRNA, rRNA, tRNA, RNAi) of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent a sense or antisense strand, to peptide nucleic acid (PNA), orto any DNA-like or RNA-like material, natural or synthetic in origin,including, e.g., RNAi (double-stranded “interfering” RNA),ribonucleoproteins (e.g., iRNPs). The term encompasses nucleic acids,i.e., oligonucleotides, containing known analogues of naturalnucleotides. The term also encompasses nucleic-acid-like structures withsynthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol.144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag(1996) Antisense Nucleic Acid Drug Dev 6:153-156.

As used herein, the term “promoter” includes all sequences capable ofdriving transcription of a coding sequence in a cell, e.g., a plantcell. Thus, promoters used in the constructs of the invention includecis-acting transcriptional control elements and regulatory sequencesthat are involved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription. “Constitutive” promoters are those that drive expressioncontinuously under most environmental conditions and states ofdevelopment or cell differentiation. “Inducible” or “regulatable”promoters direct expression of the nucleic acid of the invention underthe influence of environmental conditions or developmental conditions.Examples of environmental conditions that may affect transcription byinducible promoters include anaerobic conditions, elevated temperature,drought, or the presence of light.

“Tissue-specific” promoters are transcriptional control elements thatare only active in particular cells or tissues or organs, e.g., inplants or animals. Tissue-specific regulation may be achieved by certainintrinsic factors which ensure that genes encoding proteins specific toa given tissue are expressed. Such factors are known to exist in mammalsand plants so as to allow for specific tissues to develop.

The term “plant” includes whole plants, plant parts (e.g., leaves,stems, flowers, roots, etc.), plant protoplasts, seeds and plant cellsand progeny of same. The class of plants which can be used in the methodof the invention is generally as broad as the class of higher plantsamenable to transformation techniques, including angiosperms(monocotyledonous and dicotyledonous plants), as well as gymnosperms. Itincludes plants of a variety of ploidy levels, including polyploid,diploid, haploid and hemizygous states. As used herein, the term“transgenic plant” includes plants or plant cells into which aheterologous nucleic acid sequence has been inserted, e.g., the nucleicacids and various recombinant constructs (e.g., expression cassettes) ofthe invention.

“Amino acid” or “amino acid sequence” can include an oligopeptide,peptide, polypeptide, or protein sequence, or to a fragment, portion, orsubunit of any of these, and to naturally occurring or syntheticmolecules.

The terms “polypeptide” and “protein” can include amino acids joined toeach other by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain modified amino acids other than the 20gene-encoded amino acids. The term “polypeptide” also includes peptidesand polypeptide fragments, motifs and the like. The term also includesglycosylated polypeptides. The peptides and polypeptides of theinvention also include all “mimetic” and “peptidomimetic” forms, asdescribed in further detail, below.

The term “isolated” can mean that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.As used herein, an isolated material or composition can also be a“purified” composition, i.e., it does not require absolute purity;rather, it is intended as a relative definition. Individual nucleicacids obtained from a library can be conventionally purified toelectrophoretic homogeneity. In alternative aspects, the inventionprovides nucleic acids which have been purified from genomic DNA or fromother sequences in a library or other environment by at least one, two,three, four, five or more orders of magnitude.

The term “recombinant” can mean that the nucleic acid is adjacent to a“backbone” nucleic acid to which it is not adjacent in its naturalenvironment. In one aspect, nucleic acids represent 5% or more of thenumber of nucleic acid inserts in a population of nucleic acid “backbonemolecules.” “Backbone molecules” according to the invention includenucleic acids such as expression vectors, self-replicating nucleicacids, viruses, integrating nucleic acids, and other vectors or nucleicacids used to maintain or manipulate a nucleic acid insert of interest.In one aspect, the enriched nucleic acids represent 15%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or more of the number of nucleic acid inserts inthe population of recombinant backbone molecules.

“Recombinant” polypeptides or proteins refer to polypeptides or proteinsproduced by recombinant DNA techniques; e.g., produced from cellstransformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or protein are thoseprepared by chemical synthesis, as described in further detail, below.

A promoter sequence can be “operably linked to” a coding sequence whenRNA polymerase which initiates transcription at the promoter willtranscribe the coding sequence into mRNA, as discussed further, below.

“Oligonucleotide” can include either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides, can refer to two or more sequences that have, e.g., atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, nucleotide oramino acid residue (sequence) identity, when compared and aligned formaximum correspondence, as measured using one any known sequencecomparison algorithm, as discussed in detail below, or by visualinspection. In alternative aspects, the invention provides nucleic acidand polypeptide sequences having substantial identity to a nucleic acidof the invention, e.g., an exemplary sequence of the invention, over aregion of at least about 10, 20, 30, 40, 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000residues, or a region ranging from between about 50 residues to the fulllength of the nucleic acid or polypeptide. Nucleic acid sequences of theinvention can be substantially identical over the entire length of apolypeptide coding region.

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site of the molecule, and provided that thepolypeptide essentially retains its functional properties. Aconservative amino acid substitution, for example, substitutes one aminoacid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid or glutamine for asparagine). One or more amino acids canbe deleted, for example, from a hydrolase, resulting in modification ofthe structure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for hydrolase activity can be removed.

“Hybridization” refers to the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Stringent conditions can be defined by, for example,the concentrations of salt or formamide in the prehybridization andhybridization solutions, or by the hybridization temperature, and arewell known in the art. For example, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature, altering the timeof hybridization, as described in detail, below. In alternative aspects,nucleic acids of the invention are defined by their ability to hybridizeunder various stringency conditions (e.g., high, medium, and low), asset forth herein.

The term “variant” can include polynucleotides or polypeptides of theinvention modified at one or more base pairs, codons, introns, exons, oramino acid residues (respectively) yet still retain the biologicalactivity of a hydrolase of the invention. Variants can be produced byany number of means included methods such as, for example, error-pronePCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR,sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis,recursive ensemble mutagenesis, exponential ensemble mutagenesis,site-specific mutagenesis, gene reassembly, GSSM and any combinationthereof. Techniques for producing variant hydrolases having activity ata pH or temperature, for example, that is different from a wild-typehydrolase, are included herein.

The term “saturation mutagenesis” or “GSSM” includes a method that usesdegenerate oligonucleotide primers to introduce point mutations into apolynucleotide, as described in detail, below. The term “optimizeddirected evolution system” or “optimized directed evolution” includes amethod for reassembling fragments of related nucleic acid sequences,e.g., related genes, and explained in detail, below. The term “syntheticligation reassembly” or “SLR” includes a method of ligatingoligonucleotide fragments in a non-stochastic fashion, and explained indetail, below.

The term “syrup” can be defined as an aqueous solution or slurrycomprising carbohydrates such as mono-, oligo- or polysaccharides.

Generating and Manipulating Nucleic Acids

The invention provides nucleic acids, including expression cassettessuch as expression vectors, encoding the polypeptides (e.g., hydrolases,antibodies) of the invention. The invention also includes methods fordiscovering new hydrolase sequences using the nucleic acids of theinvention. Also provided are methods for modifying the nucleic acids ofthe invention by, e.g., synthetic ligation reassembly, optimizeddirected evolution system and/or saturation mutagenesis.

The nucleic acids of the invention can be made, isolated and/ormanipulated by, e.g., cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like. Inpracticing the methods of the invention, homologous genes can bemodified by manipulating a template nucleic acid, as described herein.The invention can be practiced in conjunction with any method orprotocol or device known in the art, which are well described in thescientific and patent literature.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides generated from these nucleic acids can beindividually isolated or cloned and tested for a desired activity. Anyrecombinant expression system can be used, including bacterial,mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA, iRNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.

Promoters suitable for expressing a polypeptide in bacteria include theE. coli lac or trp promoters, the lad promoter, the lacZ promoter, theT3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter,the lambda PL promoter, promoters from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), and the acidphosphatase promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, heat shock promoters,the early and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused.

Tissue-Specific Plant Promoters

The invention provides expression cassettes that can be expressed in atissue-specific manner, e.g., that can express a hydrolase of theinvention in a tissue-specific manner. The invention also providesplants or seeds that express a hydrolase of the invention in atissue-specific manner. The tissue-specificity can be seed specific,stem specific, leaf specific, root specific, fruit specific and thelike.

In one aspect, a constitutive promoter such as the CaMV 35S promoter canbe used for expression in specific parts of the plant or seed orthroughout the plant. For example, for overexpression of a hydrolase ofthe invention, a plant promoter fragment can be employed which willdirect expression of a nucleic acid in some or all tissues of a plant,e.g., a regenerated plant. Such “constitutive” promoters and are activeunder most environmental conditions and states of development or celldifferentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, andother transcription initiation regions from various plant genes known tothose of skill. Such genes include, e.g., ACT11 from Arabidopsis (Huang(1996) Plant Mol. Biol. 33:125-139); Cat3 from Arabidopsis (GenBank No.U43147, Zhong (1996) Mol. Gen. Genet. 251:196-203); the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankNo. X74782, Solocombe (1994) Plant Physiol. 104:1167-1176); GPc1 frommaize (GenBank No. X15596; Martinez (1989) J. Mol. Biol 208:551-565);the Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant Mol.Biol. 33:97-112); plant promoters described in U.S. Pat. Nos. 4,962,028;5,633,440.

The invention uses tissue-specific or constitutive promoters derivedfrom viruses which can include, e.g., the tobamovirus subgenomicpromoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; therice tungro bacilliform virus (RTBV), which replicates only in phloemcells in infected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassava vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

Alternatively, the plant promoter may direct expression of ahydrolase-expressing nucleic acid in a specific tissue, organ or celltype (i.e. tissue-specific promoters) or may be otherwise under moreprecise environmental or developmental control or under the control ofan inducible promoter. Examples of environmental conditions that mayaffect transcription include anaerobic conditions, elevated temperature,the presence of light, or sprayed with chemicals/hormones. For example,the invention incorporates the drought-inducible promoter of maize (Busk(1997) supra); the cold, drought, and high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897 909).

Tissue-specific promoters can promote transcription only within acertain time frame of developmental stage within that tissue. See, e.g.,Blazquez (1998) Plant Cell 10:791-800, characterizing the ArabidopsisLEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77,describing the transcription factor SPL3, which recognizes a conservedsequence motif in the promoter region of the A. thaliana floral meristemidentity gene AP1; and Mandel (1995) Plant Molecular Biology, Vol. 29,pp 995-1004, describing the meristem promoter eIF4. Tissue specificpromoters which are active throughout the life cycle of a particulartissue can be used. In one aspect, the nucleic acids of the inventionare operably linked to a promoter active primarily only in cotton fibercells. In one aspect, the nucleic acids of the invention are operablylinked to a promoter active primarily during the stages of cotton fibercell elongation, e.g., as described by Rinehart (1996) supra. Thenucleic acids can be operably linked to the Fbl2A gene promoter to bepreferentially expressed in cotton fiber cells (Ibid). See also, John(1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat.Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promotersand methods for the construction of transgenic cotton plants.Root-specific promoters may also be used to express the nucleic acids ofthe invention. Examples of root-specific promoters include the promoterfrom the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol.123:39-60). Other promoters that can be used to express the nucleicacids of the invention include, e.g., ovule-specific, embryo-specific,endosperm-specific, integument-specific, seed coat-specific promoters,or some combination thereof; a leaf-specific promoter (see, e.g., Busk(1997) Plant J. 11:1285 1295, describing a leaf-specific promoter inmaize); the ORF13 promoter from Agrobacterium rhizogenes (which exhibitshigh activity in roots, see, e.g., Hansen (1997) supra); a maize pollenspecific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161168); a tomato promoter active during fruit ripening, senescence andabscission of leaves and, to a lesser extent, of flowers can be used(see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specificpromoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol.Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermaltissue of vegetative and floral shoot apices of transgenic alfalfamaking it a useful tool to target the expression of foreign genes to theepidermal layer of actively growing shoots or fibers; the ovule-specificBEL1 gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No.U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583,describing a plant promoter region is capable of conferring high levelsof transcription in meristematic tissue and/or rapidly dividing cells.

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents whichcan be applied to the plant, such as herbicides or antibiotics. Forexample, the maize In2-2 promoter, activated by benzenesulfonamideherbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.38:568-577); application of different herbicide safeners inducesdistinct gene expression patterns, including expression in the root,hydathodes, and the shoot apical meristem. Coding sequence can be underthe control of, e.g., a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324). Using chemically- (e.g., hormone- or pesticide-) inducedpromoters, i.e., promoter responsive to a chemical which can be appliedto the transgenic plant in the field, expression of a polypeptide of theinvention can be induced at a particular stage of development of theplant. Thus, the invention also provides for transgenic plantscontaining an inducible gene encoding for polypeptides of the inventionwhose host range is limited to target plant species, such as corn, rice,barley, wheat, potato or other crops, inducible at any stage ofdevelopment of the crop.

Tissue-specific plant promoters may drive expression of operably linkedsequences in tissues other than the target tissue. Thus, atissue-specific promoter is one that drives expression preferentially inthe target tissue or cell type, but may also lead to some expression inother tissues as well.

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents. Thesereagents include, e.g., herbicides, synthetic auxins, or antibioticswhich can be applied, e.g., sprayed, onto transgenic plants. Inducibleexpression of the hydrolase-producing nucleic acids of the inventionwill allow the grower to select plants with the optimal starch/sugarratio. The development of plant parts can thus controlled. In this waythe invention provides the means to facilitate the harvesting of plantsand plant parts. For example, in various embodiments, the maize In2-2promoter, activated by benzenesulfonamide herbicide safeners, is used(De Veylder (1997) Plant Cell Physiol. 38:568-577); application ofdifferent herbicide safeners induces distinct gene expression patterns,including expression in the root, hydathodes, and the shoot apicalmeristem. Coding sequences of the invention are also under the controlof a tetracycline-inducible promoter, e.g., as described with transgenictobacco plants containing the Avena sativa L. (oat) argininedecarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylicacid-responsive element (Stange (1997) Plant J. 11:1315-1324).

If proper polypeptide expression is desired, a polyadenylation region atthe 3′-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from genes in the Agrobacterial T-DNA.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding thehydrolases and antibodies of the invention. Expression vectors andcloning vehicles of the invention can comprise viral particles,baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterialartificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul poxvirus, pseudorabies and derivatives of SV40), P1-based artificialchromosomes, yeast plasmids, yeast artificial chromosomes, and any othervectors specific for specific hosts of interest (such as bacillus,Aspergillus and yeast). Vectors of the invention can includechromosomal, non-chromosomal and synthetic DNA sequences. Large numbersof suitable vectors are known to those of skill in the art, and arecommercially available. Exemplary vectors are include: bacterial: pQEvectors (Qiagen), pBluescript plasmids, pNH vectors, (lambda-ZAP vectors(Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic:pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia).However, any other plasmid or other vector may be used so long as theyare replicable and viable in the host. Low copy number or high copynumber vectors may be employed with the present invention.

The expression vector may comprise a promoter, a ribosome binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP1 gene. Promoter regions can be selected from anydesired gene using chloramphenicol transferase (CAT) vectors or othervectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin by 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A DNA sequence may be inserted into a vector by a variety of procedures.In general, the DNA sequence is ligated to the desired position in thevector following digestion of the insert and the vector with appropriaterestriction endonucleases. Alternatively, blunt ends in both the insertand the vector may be ligated. A variety of cloning techniques are knownin the art, e.g., as described in Ausubel and Sambrook. Such proceduresand others are deemed to be within the scope of those skilled in theart.

The vector may be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which may be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

The nucleic acids of the invention can be expressed in expressioncassettes, vectors or viruses and transiently or stably expressed inplant cells and seeds. One exemplary transient expression system usesepisomal expression systems, e.g., cauliflower mosaic virus (CaMV) viralRNA generated in the nucleus by transcription of an episomalmini-chromosome containing supercoiled DNA, see, e.g., Covey (1990)Proc. Natl. Acad. Sci. USA 87:1633-1637. Alternatively, codingsequences, i.e., all or sub-fragments of sequences of the invention canbe inserted into a plant host cell genome becoming an integral part ofthe host chromosomal DNA. Sense or antisense transcripts can beexpressed in this manner. A vector comprising the sequences (e.g.,promoters or coding regions) from nucleic acids of the invention cancomprise a marker gene that confers a selectable phenotype on a plantcell or a seed. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosulfuron or Basta.

Expression vectors capable of expressing nucleic acids and proteins inplants are well known in the art, and can include, e.g., vectors fromAgrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J.16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993)Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g.,Cecchini (1997) Mol. Plant Microbe Interact. 10:1094-1101), maize Ac/Dstransposable element (see, e.g., Rubin (1997) Mol. Cell. Biol.17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194),and the maize suppressor-mutator (Spm) transposable element (see, e.g.,Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.

In one aspect, the expression vector can have two replication systems toallow it to be maintained in two organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector can contain at least one sequence homologous to thehost cell genome. It can contain two homologous sequences which flankthe expression construct. The integrating vector can be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

Expression vectors of the invention may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed, e.g., genes which render the bacteria resistant to drugssuch as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycinand tetracycline. Selectable markers can also include biosyntheticgenes, such as those in the histidine, tryptophan and leucinebiosynthetic pathways.

Host Cells and Transformed Cells

The invention also provides a transformed cell comprising a nucleic acidsequence of the invention, e.g., a sequence encoding a hydrolase or anantibody of the invention, or a vector of the invention. The host cellmay be any of the host cells familiar to those skilled in the art,including prokaryotic cells, eukaryotic cells, such as bacterial cells,fungal cells, yeast cells, mammalian cells, insect cells, or plantcells. Enzymes of the invention can be expressed in any host cell, e.g.,any bacterial cell, any yeast cell, e.g., Pichia pastoris, Saccharomycescerevisiae or Schizosaccharomyces pombe. Exemplary bacterial cellsinclude E. coli, Lactococcus lactis, Streptomyces, Bacillus subtilis,Bacillus cereus, Salmonella typhimurium or any species within the generaBacillus, Streptomyces and Staphylococcus. Exemplary insect cellsinclude Drosophila S2 and Spodoptera Sf9. Exemplary animal cells includeCHO, COS or Bowes melanoma or any mouse or human cell line. Theselection of an appropriate host is within the abilities of thoseskilled in the art. Techniques for transforming a wide variety of higherplant species are well known and described in the technical andscientific literature. See, e.g., Weising (1988) Ann Rev. Genet.22:421-477, U.S. Pat. No. 5,750,870.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

In one aspect, the nucleic acids or vectors of the invention areintroduced into the cells for screening, thus, the nucleic acids enterthe cells in a manner suitable for subsequent expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type. Exemplary methods include CaPO₄ precipitation, liposomefusion, lipofection (e.g., LIPOFECTIN™), electroporation, viralinfection, etc. The candidate nucleic acids may stably integrate intothe genome of the host cell (for example, with retroviral introduction)or may exist either transiently or stably in the cytoplasm (i.e. throughthe use of traditional plasmids, utilizing standard regulatorysequences, selection markers, etc.). As many pharmaceutically importantscreens require human or model mammalian cell targets, retroviralvectors capable of transfecting such targets are preferred.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts and other cell linescapable of expressing proteins from a compatible vector, such as theC127, 3T3, CHO, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids encoding the polypeptides ofthe invention, or modified nucleic acids, can be reproduced by, e.g.,amplification. The invention provides amplification primer sequencepairs for amplifying nucleic acids encoding a hydrolase, e.g., anesterase, acylase, lipase, phospholipase or protease, where the primerpairs are capable of amplifying nucleic acid sequences including theexemplary SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ IDNO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ IDNO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173,SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ IDNO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201,SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ IDNO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229,SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ IDNO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257,SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ IDNO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285,SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ IDNO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313,SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ IDNO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQID NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQ ID NO:339, SEQ ID NO:341,SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:349, SEQ IDNO:351, SEQ ID NO:353, SEQ ID NO:355, SEQ ID NO:357, SEQ ID NO:359, SEQID NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:369,SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:375, SEQ ID NO:377, SEQ IDNO:379, SEQ ID NO:381, SEQ ID NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQID NO:389, SEQ ID NO:391, SEQ ID NO:393, SEQ ID NO:395, SEQ ID NO:397,SEQ ID NO:399, SEQ ID NO:401, SEQ ID NO:403, SEQ ID NO:405, SEQ IDNO:407, SEQ ID NO:409, SEQ ID NO:411, SEQ ID NO:413, SEQ ID NO:415, SEQID NO:417, SEQ ID NO:419, SEQ ID NO:421, SEQ ID NO:423, SEQ ID NO:425,SEQ ID NO:427, SEQ ID NO:429, SEQ ID NO:431, SEQ ID NO:433, SEQ IDNO:435, SEQ ID NO:437, SEQ ID NO:439, SEQ ID NO:441, SEQ ID NO:443, SEQID NO:445, SEQ ID NO:447, SEQ ID NO:449, SEQ ID NO:451, SEQ ID NO:453,SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459, SEQ ID NO:461, SEQ IDNO:463, SEQ ID NO:465, SEQ ID NO:467, SEQ ID NO:469, SEQ ID NO:471, SEQID NO:473, SEQ ID NO:475, SEQ ID NO:477, SEQ ID NO:479, SEQ ID NO:481,SEQ ID NO:483, SEQ ID NO:485, SEQ ID NO:487, SEQ ID NO:489, SEQ IDNO:491, SEQ ID NO:493, SEQ ID NO:495, SEQ ID NO:497, SEQ ID NO:499, SEQID NO:501, SEQ ID NO:503, SEQ ID NO:505, SEQ ID NO:507, SEQ ID NO:509,SEQ ID NO:511, SEQ ID NO:513, SEQ ID NO:515, SEQ ID NO:517, SEQ IDNO:519, SEQ ID NO:521, SEQ ID NO:523, SEQ ID NO:525, SEQ ID NO:527, SEQID NO:529, SEQ ID NO:531, SEQ ID NO:533, SEQ ID NO:535, SEQ ID NO:537,SEQ ID NO:539, SEQ ID NO:541, SEQ ID NO:543, SEQ ID NO:545, SEQ IDNO:547, SEQ ID NO:549, SEQ ID NO:551, SEQ ID NO:553, SEQ ID NO:555, SEQID NO:557, SEQ ID NO:559, SEQ ID NO:561, SEQ ID NO:563, SEQ ID NO:565,SEQ ID NO:567, SEQ ID NO:569, SEQ ID NO:571, SEQ ID NO:573, SEQ IDNO:575, SEQ ID NO:577, SEQ ID NO:579, SEQ ID NO:581, SEQ ID NO:583, SEQID NO:585, SEQ ID NO:587, SEQ ID NO:589, SEQ ID NO:591, SEQ ID NO:593,SEQ ID NO:595, SEQ ID NO:597, SEQ ID NO:599, SEQ ID NO:601, SEQ IDNO:603, SEQ ID NO:605, SEQ ID NO:607, SEQ ID NO:609, SEQ ID NO:611, SEQID NO:613, SEQ ID NO:615, SEQ ID NO:617, SEQ ID NO:619, SEQ ID NO:621,SEQ ID NO:623, SEQ ID NO:625, SEQ ID NO:627, SEQ ID NO:629, SEQ IDNO:631, SEQ ID NO:633, SEQ ID NO:635, SEQ ID NO:637, SEQ ID NO:639, SEQID NO:641, SEQ ID NO:643, SEQ ID NO:645, SEQ ID NO:647, SEQ ID NO:649,SEQ ID NO:651, SEQ ID NO:653, SEQ ID NO:655, SEQ ID NO:657, SEQ IDNO:659, SEQ ID NO:661, SEQ ID NO:663, SEQ ID NO:665, SEQ ID NO:667, SEQID NO:669, SEQ ID NO:671, SEQ ID NO:673, SEQ ID NO:675, SEQ ID NO:677,SEQ ID NO:679, SEQ ID NO:681, SEQ ID NO:683, SEQ ID NO:685, SEQ IDNO:687, SEQ ID NO:689, SEQ ID NO:691, SEQ ID NO:693, SEQ ID NO:695, SEQID NO:697, SEQ ID NO:699, SEQ ID NO:701, SEQ ID NO:703, SEQ ID NO:705,SEQ ID NO:707, SEQ ID NO:709, SEQ ID NO:711, SEQ ID NO:713, SEQ IDNO:715, SEQ ID NO:717, SEQ ID NO:719, SEQ ID NO:721, SEQ ID NO:723, SEQID NO:725, SEQ ID NO:727, SEQ ID NO:729, SEQ ID NO:731, SEQ ID NO:733,SEQ ID NO:735, SEQ ID NO:737, SEQ ID NO:739, SEQ ID NO:741, SEQ IDNO:743, SEQ ID NO:745, SEQ ID NO:747, SEQ ID NO:749, SEQ ID NO:751, SEQID NO:753, SEQ ID NO:755, SEQ ID NO:757, SEQ ID NO:759, SEQ ID NO:761,SEQ ID NO:763, SEQ ID NO:765, SEQ ID NO:767, SEQ ID NO:769, SEQ IDNO:771, SEQ ID NO:773, SEQ ID NO:775, SEQ ID NO:777, SEQ ID NO:779, SEQID NO:781, SEQ ID NO:783, SEQ ID NO:785, SEQ ID NO:787, SEQ ID NO:789,SEQ ID NO:791, SEQ ID NO:793, SEQ ID NO:795, SEQ ID NO:797, SEQ IDNO:799, SEQ ID NO:801, SEQ ID NO:803, SEQ ID NO:805, SEQ ID NO:807, SEQID NO:809, SEQ ID NO:811, SEQ ID NO:813, SEQ ID NO:815, SEQ ID NO:817,SEQ ID NO:819, SEQ ID NO:821, SEQ ID NO:823, SEQ ID NO:825, SEQ IDNO:827, SEQ ID NO:829, SEQ ID NO:831, SEQ ID NO:833, SEQ ID NO:835, SEQID NO:837, SEQ ID NO:839, SEQ ID NO:841, SEQ ID NO:843, SEQ ID NO:845,SEQ ID NO:847, SEQ ID NO:849, SEQ ID NO:851, SEQ ID NO:853, SEQ IDNO:855, SEQ ID NO:857, SEQ ID NO:859, SEQ ID NO:861, SEQ ID NO:863, SEQID NO:865, SEQ ID NO:867, SEQ ID NO:869, SEQ ID NO:871, SEQ ID NO:873,SEQ ID NO:875, SEQ ID NO:877, SEQ ID NO:879, SEQ ID NO:881, SEQ IDNO:883, SEQ ID NO:885, SEQ ID NO:887, SEQ ID NO:889, SEQ ID NO:891, SEQID NO:893, SEQ ID NO:895, SEQ ID NO:897, SEQ ID NO:899, SEQ ID NO:901,SEQ ID NO:903, SEQ ID NO:905, SEQ ID NO:907, SEQ ID NO:909, SEQ IDNO:911, SEQ ID NO:913, SEQ ID NO:915, SEQ ID NO:917, SEQ ID NO:919, SEQID NO:921, SEQ ID NO:923, SEQ ID NO:925, SEQ ID NO:927, SEQ ID NO:929,SEQ ID NO:931, SEQ ID NO:933, SEQ ID NO:935, SEQ ID NO:937, SEQ IDNO:939, SEQ ID NO:941, SEQ ID NO:943, SEQ ID NO:945, SEQ ID NO:947, SEQID NO:949, SEQ ID NO:951 or SEQ ID NO:953. One of skill in the art candesign amplification primer sequence pairs for any part of or the fulllength of these sequences.

Additionally, the invention provides amplification primer sequence pairsfor amplifying nucleic acids encoding a hydrolase, e.g., an esterase,acylase, lipase, phospholipase or protease, where the primer pairs arecapable of amplifying nucleic acid sequences including the exemplary SEQID NO:955, SEQ ID NO:957, SEQ ID NO:959, SEQ ID NO:961, SEQ ID NO:963,SEQ ID NO:965, SEQ ID NO:967, SEQ ID NO:969, SEQ ID NO:971, SEQ IDNO:973, SEQ ID NO:975, SEQ ID NO:977, SEQ ID NO:979, SEQ ID NO:981, orSEQ ID NO:983.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified. The skilledartisan can select and design suitable oligonucleotide amplificationprimers. Amplification methods are also well known in the art, andinclude, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS,A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y.(1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y.,ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560;Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

The invention also provides amplification primer pairs comprisingsequences of the invention, for example, wherein the primer paircomprises a first member having a sequence as set forth by about thefirst (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or moreresidues of a nucleic acid of the invention, and a second member havinga sequence as set forth by about the first (the 5′) 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39 or 40 or more residues of the complementary strand ofthe first member.

Determining the Degree of Sequence Identity

The invention provides nucleic acids having at least nucleic acid, orcomplete (100%) sequence identity to a nucleic acid of the invention,e.g., an exemplary nucleic acid of the invention (e.g., having asequence as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, etc.); and polypeptides having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to a polypeptide of the invention, e.g., an exemplarypolypeptide having a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, etc. Inalternative aspects, the sequence identity can be over a region of atleast about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or moreconsecutive residues, or the full length of the nucleic acid orpolypeptide. The extent of sequence identity (homology) may bedetermined using any computer program and associated parameters,including those described herein, such as BLAST 2.2.2. or FASTA version3.0t78, with the default parameters.

FIG. 30 is a chart describing selected characteristics of exemplarynucleic acids and polypeptides of the invention, including sequenceidentity comparison of the exemplary sequences to public databases. Allsequences described in FIG. 30 have been subject to a BLAST search (asdescribed in detail, below) against two sets of databases. The firstdatabase set is available through NCBI (National Center forBiotechnology Information). All results from searches against thesedatabases are found in the columns entitled “NR Description”, “NRAccession Code”, “NR Evalue” or “NR Organism”. “NR” refers to theNon-Redundant nucleotide database maintained by NCBI. This database is acomposite of GenBank, GenBank updates, and EMBL updates. The entries inthe column “NR Description” refer to the definition line in any givenNCBI record, which includes a description of the sequence, such as thesource organism, gene name/protein name, or some description of thefunction of the sequence. The entries in the column “NR Accession Code”refer to the unique identifier given to a sequence record. The entriesin the column “NR Evalue” refer to the Expect value (Evalue), whichrepresents the probability that an alignment score as good as the onefound between the query sequence (the sequences of the invention) and adatabase sequence would be found in the same number of comparisonsbetween random sequences as was done in the present BLAST search. Theentries in the column “NR Organism” refer to the source organism of thesequence identified as the closest BLAST hit. The second set ofdatabases is collectively known as the Geneseq database, which isavailable through Thomson Derwent (Philadelphia, Pa.). All results fromsearches against this database are found in the columns entitled“Geneseq Protein Description”, “Geneseq Protein Accession Code”,“Geneseq Protein Evalue”, “Geneseq DNA Description”, “Geneseq DNAAccession Code” or “Geneseq DNA Evalue”. The information found in thesecolumns is comparable to the information found in the NR columnsdescribed above, except that it was derived from BLAST searches againstthe Geneseq™ database instead of the NCBI databases. In addition, thistable includes the column “Predicted EC No.”. An EC number is the numberassigned to a type of enzyme according to a scheme of standardizedenzyme nomenclature developed by the Enzyme Commission of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (IUBMB). The results in the “Predicted EC No.” columnare determined by a BLAST search against the Kegg (Kyoto Encyclopedia ofGenes and Genomes) database. If the top BLAST match has an Evalue equalto or less than e⁻⁶, the EC number assigned to the top match is enteredinto the table. The EC number of the top hit is used as a guide to whatthe EC number of the sequence of the invention might be. The columns“Query DNA Length” and “Query Protein Length” refer to the number ofnucleotides or the number amino acids, respectively, in the sequence ofthe invention that was searched or queried against either the NCBI orGeneseq databases. The columns “Geneseq or NR DNA Length” and “Geneseqor NR Protein Length” refer to the number of nucleotides or the numberamino acids, respectively, in the sequence of the top match from theBLAST search. The results provided in these columns are from the searchthat returned the lower Evalue, either from the NCBI databases or theGeneseq database. The columns “Geneseq or NR % ID Protein” and “Geneseqor NR % ID DNA” refer to the percent sequence identity between thesequence of the invention and the sequence of the top BLAST match. Theresults provided in these columns are from the search that returned thelower Evalue, either from the NCBI databases or the Geneseq database.

Homologous sequences also include RNA sequences in which uridinesreplace the thymines in the nucleic acid sequences. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. It will beappreciated that the nucleic acid sequences as set forth herein can berepresented in the traditional single character format (see, e.g.,Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co., New York) orin any other format which records the identity of the nucleotides in asequence.

Various sequence comparison programs identified herein are used in thisaspect of the invention. Protein and/or nucleic acid sequence identities(homologies) may be evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are not limited to, TBLASTN, BLASTP, FASTA,TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410,1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higginset al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol.Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272,1993).

Homology or identity can be measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection. For sequencecomparison, one sequence can act as a reference sequence (e.g., anexemplary nucleic acid or polypeptide sequence of the invention) towhich test sequences are compared. When using a sequence comparisonalgorithm, test and reference sequences are entered into a computer,subsequence coordinates are designated, if necessary, and sequencealgorithm program parameters are designated. Default program parameterscan be used, or alternative parameters can be designated. The sequencecomparison algorithm then calculates the percent sequence identities forthe test sequences relative to the reference sequence, based on theprogram parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the numbers of contiguous residues. For example, inalternative aspects of the invention, continugous residues ranginganywhere from 20 to the full length of an exemplary polypeptide ornucleic acid sequence of the invention, are compared to a referencesequence of the same number of contiguous positions after the twosequences are optimally aligned. If the reference sequence has therequisite sequence identity to an exemplary polypeptide or nucleic acidsequence of the invention, e.g., in alternative aspects, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto an exemplary polypeptide or nucleic acid sequence of the invention,that sequence is within the scope of the invention. In alternativeembodiments, subsequences ranging from about 20 to 600, about 50 to 200,and about 100 to 150 are compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. Methods of alignment of sequence for comparison are well knownin the art. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search forsimilarity method of person & Lipman, Proc. Nat'l. Acad. Sci. USA85:2444, 1988, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection. Other algorithms for determininghomology or identity include, for example, in addition to a BLASTprogram (Basic Local Alignment Search Tool at the National Center forBiological Information), ALIGN, AMAS (Analysis of Multiply AlignedSequences), AMPS (Protein Multiple Sequence Alignment), ASSET (AlignedSegment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project(Gibbs, 1995). Several genomes have been sequenced, e.g., M. genitalium(Fraser et al., 1995), M. jannaschii (Bult et al., 1996), H. influenzae(Fleischmann et al., 1995), E. coli (Blattner et al., 1997), and yeast(S. cerevisiae) (Mewes et al., 1997), and D. melanogaster (Adams et al.,2000). Significant progress has also been made in sequencing the genomesof model organism, such as mouse, C. elegans, and Arabadopsis sp.Databases containing genomic information annotated with some functionalinformation are maintained by different organization, and are accessiblevia the internet.

BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practicethe invention. They are described, e.g., in Altschul (1977) Nuc. AcidsRes. 25:3389-3402; Altschul (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul (1990) supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. The BLAST algorithm also performs a statisticalanalysis of the similarity between two sequences (see, e.g., Karlin &Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873). One measure ofsimilarity provided by BLAST algorithm is the smallest sum probability(P(N)), which provides an indication of the probability by which a matchbetween two nucleotide or amino acid sequences would occur by chance.For example, a nucleic acid is considered similar to a referencessequence if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.2, morepreferably less than about 0.01, and most preferably less than about0.001. In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”). Forexample, five specific BLAST programs can be used to perform thefollowing task: (1) BLASTP and BLAST3 compare an amino acid querysequence against a protein sequence database; (2) BLASTN compares anucleotide query sequence against a nucleotide sequence database; (3)BLASTX compares the six-frame conceptual translation products of a querynucleotide sequence (both strands) against a protein sequence database;(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and, (5) TBLASTX compares the six-frame translations of a nucleotidequery sequence against the six-frame translations of a nucleotidesequence database. The BLAST programs identify homologous sequences byidentifying similar segments, which are referred to herein as“high-scoring segment pairs,” between a query amino or nucleic acidsequence and a test sequence which is preferably obtained from a proteinor nucleic acid sequence database. High-scoring segment pairs arepreferably identified (i.e., aligned) by means of a scoring matrix, manyof which are known in the art. Preferably, the scoring matrix used isthe BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992;Henikoff and Henikoff, Proteins 17:49-61, 1993). Less preferably, thePAM or PAM250 matrices may also be used (see, e.g., Schwartz andDayhoff, eds., 1978, Matrices for Detecting Distance Relationships:Atlas of Protein Sequence and Structure, Washington: National BiomedicalResearch Foundation).

In one aspect of the invention, to determine if a nucleic acid has therequisite sequence identity to be within the scope of the invention, theNCBI BLAST 2.2.2 programs is used, default options to blastp. There areabout 38 setting options in the BLAST 2.2.2 program. In this exemplaryaspect of the invention, all default values are used except for thedefault filtering setting (i.e., all parameters set to default exceptfiltering which is set to OFF); in its place a “−F F” setting is used,which disables filtering. Use of default filtering often results inKarlin-Altschul violations due to short length of sequence.

The default values used in this exemplary aspect of the invention, andto determine the values in FIG. 30, as discussed above, include:

“Filter for low complexity: ON

Word Size: 3

Matrix: Blosum62

Gap Costs: Existence:11

Extension:1”

Other default settings are: filter for low complexity OFF, word size of3 for protein, BLOSUM62 matrix, gap existence penalty of −11 and a gapextension penalty of −1. An exemplary NCBI BLAST 2.2.2 program settingis set forth in Example 1, below. Note that the “−W” option defaults to0. This means that, if not set, the word size defaults to 3 for proteinsand 11 for nucleotides.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies,motifs and the like in silico, the sequence of the invention can bestored, recorded, and manipulated on any medium which can be read andaccessed by a computer.

Accordingly, the invention provides computers, computer systems,computer readable mediums, computer programs products and the likerecorded or stored thereon the nucleic acid and polypeptide sequences ofthe invention. As used herein, the words “recorded” and “stored” referto a process for storing information on a computer medium. A skilledartisan can readily adopt any known methods for recording information ona computer readable medium to generate manufactures comprising one ormore of the nucleic acid and/or polypeptide sequences of the invention.

Another aspect of the invention is a computer readable medium havingrecorded thereon at least one nucleic acid and/or polypeptide sequenceof the invention. Computer readable media include magnetically readablemedia, optically readable media, electronically readable media andmagnetic/optical media. For example, the computer readable media may bea hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital VersatileDisk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) aswell as other types of other media known to those skilled in the art.

Aspects of the invention include systems (e.g., internet based systems),particularly computer systems, which store and manipulate the sequencesand sequence information described herein. One example of a computersystem 100 is illustrated in block diagram form in FIG. 1. As usedherein, “a computer system” refers to the hardware components, softwarecomponents, and data storage components used to analyze a nucleotide orpolypeptide sequence of the invention. The computer system 100 caninclude a processor for processing, accessing and manipulating thesequence data. The processor 105 can be any well-known type of centralprocessing unit, such as, for example, the Pentium III from IntelCorporation, or similar processor from Sun, Motorola, Compaq, AMD orInternational Business Machines. The computer system 100 is a generalpurpose system that comprises the processor 105 and one or more internaldata storage components 110 for storing data, and one or more dataretrieving devices for retrieving the data stored on the data storagecomponents. A skilled artisan can readily appreciate that any one of thecurrently available computer systems are suitable.

In one aspect, the computer system 100 includes a processor 105connected to a bus which is connected to a main memory 115 (preferablyimplemented as RAM) and one or more internal data storage devices 110,such as a hard drive and/or other computer readable media having datarecorded thereon. The computer system 100 can further include one ormore data retrieving device 118 for reading the data stored on theinternal data storage devices 110. The data retrieving device 118 mayrepresent, for example, a floppy disk drive, a compact disk drive, amagnetic tape drive, or a modem capable of connection to a remote datastorage system (e.g., via the internet) etc. In some embodiments, theinternal data storage device 110 is a removable computer readable mediumsuch as a floppy disk, a compact disk, a magnetic tape, etc. containingcontrol logic and/or data recorded thereon. The computer system 100 mayadvantageously include or be programmed by appropriate software forreading the control logic and/or the data from the data storagecomponent once inserted in the data retrieving device. The computersystem 100 includes a display 120 which is used to display output to acomputer user. It should also be noted that the computer system 100 canbe linked to other computer systems 125 a-c in a network or wide areanetwork to provide centralized access to the computer system 100.Software for accessing and processing the nucleotide or amino acidsequences of the invention can reside in main memory 115 duringexecution. In some aspects, the computer system 100 may further comprisea sequence comparison algorithm for comparing a nucleic acid sequence ofthe invention. The algorithm and sequence(s) can be stored on a computerreadable medium. A “sequence comparison algorithm” refers to one or moreprograms which are implemented (locally or remotely) on the computersystem 100 to compare a nucleotide sequence with other nucleotidesequences and/or compounds stored within a data storage means. Forexample, the sequence comparison algorithm may compare the nucleotidesequences of the invention stored on a computer readable medium toreference sequences stored on a computer readable medium to identifyhomologies or structural motifs.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In some aspects,the parameters may be the default parameters used by the algorithms inthe absence of instructions from the user. FIG. 2 is a flow diagramillustrating one aspect of a process 200 for comparing a new nucleotideor protein sequence with a database of sequences in order to determinethe homology levels between the new sequence and the sequences in thedatabase. The database of sequences can be a private database storedwithin the computer system 100, or a public database such as GENBANKthat is available through the Internet. The process 200 begins at astart state 201 and then moves to a state 202 wherein the new sequenceto be compared is stored to a memory in a computer system 100. Asdiscussed above, the memory could be any type of memory, including RAMor an internal storage device. The process 200 then moves to a state 204wherein a database of sequences is opened for analysis and comparison.The process 200 then moves to a state 206 wherein the first sequencestored in the database is read into a memory on the computer. Acomparison is then performed at a state 210 to determine if the firstsequence is the same as the second sequence. It is important to notethat this step is not limited to performing an exact comparison betweenthe new sequence and the first sequence in the database. Well-knownmethods are known to those of skill in the art for comparing twonucleotide or protein sequences, even if they are not identical. Forexample, gaps can be introduced into one sequence in order to raise thehomology level between the two tested sequences. The parameters thatcontrol whether gaps or other features are introduced into a sequenceduring comparison are normally entered by the user of the computersystem. Once a comparison of the two sequences has been performed at thestate 210, a determination is made at a decision state 210 whether thetwo sequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200. If a determination is made that the two sequences are thesame, the process 200 moves to a state 214 wherein the name of thesequence from the database is displayed to the user. This state notifiesthe user that the sequence with the displayed name fulfills the homologyconstraints that were entered. Once the name of the stored sequence isdisplayed to the user, the process 200 moves to a decision state 218wherein a determination is made whether more sequences exist in thedatabase. If no more sequences exist in the database, then the process200 terminates at an end state 220. However, if more sequences do existin the database, then the process 200 moves to a state 224 wherein apointer is moved to the next sequence in the database so that it can becompared to the new sequence. In this manner, the new sequence isaligned and compared with every sequence in the database. It should benoted that if a determination had been made at the decision state 212that the sequences were not homologous, then the process 200 would moveimmediately to the decision state 218 in order to determine if any othersequences were available in the database for comparison. Accordingly,one aspect of the invention is a computer system comprising a processor,a data storage device having stored thereon a nucleic acid sequence ofthe invention and a sequence comparer for conducting the comparison. Thesequence comparer may indicate a homology level between the sequencescompared or identify structural motifs, or it may identify structuralmotifs in sequences which are compared to these nucleic acid codes andpolypeptide codes. FIG. 3 is a flow diagram illustrating one embodimentof a process 250 in a computer for determining whether two sequences arehomologous. The process 250 begins at a start state 252 and then movesto a state 254 wherein a first sequence to be compared is stored to amemory. The second sequence to be compared is then stored to a memory ata state 256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it can be a single letter amino acid code so thatthe first and sequence sequences can be easily compared. A determinationis then made at a decision state 264 whether the two characters are thesame. If they are the same, then the process 250 moves to a state 268wherein the next characters in the first and second sequences are read.A determination is then made whether the next characters are the same.If they are, then the process 250 continues this loop until twocharacters are not the same. If a determination is made that the nexttwo characters are not the same, the process 250 moves to a decisionstate 274 to determine whether there are any more characters eithersequence to read. If there are not any more characters to read, then theprocess 250 moves to a state 276 wherein the level of homology betweenthe first and second sequences is displayed to the user. The level ofhomology is determined by calculating the proportion of charactersbetween the sequences that were the same out of the total number ofsequences in the first sequence. Thus, if every character in a first 100nucleotide sequence aligned with an every character in a secondsequence, the homology level would be 100%.

Alternatively, the computer program can compare a reference sequence toa sequence of the invention to determine whether the sequences differ atone or more positions. The program can record the length and identity ofinserted, deleted or substituted nucleotides or amino acid residues withrespect to the sequence of either the reference or the invention. Thecomputer program may be a program which determines whether a referencesequence contains a single nucleotide polymorphism (SNP) with respect toa sequence of the invention, or, whether a sequence of the inventioncomprises a SNP of a known sequence. Thus, in some aspects, the computerprogram is a program which identifies SNPs. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 3. The method can be performed by reading a sequenceof the invention and the reference sequences through the use of thecomputer program and identifying differences with the computer program.

In other aspects the computer based system comprises an identifier foridentifying features within a nucleic acid or polypeptide of theinvention. An “identifier” refers to one or more programs whichidentifies certain features within a nucleic acid sequence. For example,an identifier may comprise a program which identifies an open readingframe (ORF) in a nucleic acid sequence. FIG. 4 is a flow diagramillustrating one aspect of an identifier process 300 for detecting thepresence of a feature in a sequence. The process 300 begins at a startstate 302 and then moves to a state 304 wherein a first sequence that isto be checked for features is stored to a memory 115 in the computersystem 100. The process 300 then moves to a state 306 wherein a databaseof sequence features is opened. Such a database would include a list ofeach feature's attributes along with the name of the feature. Forexample, a feature name could be “Initiation Codon” and the attributewould be “ATG”. Another example would be the feature name “TAATAA Box”and the feature attribute would be “TAATAA”. An example of such adatabase is produced by the University of Wisconsin Genetics ComputerGroup. Alternatively, the features may be structural polypeptide motifssuch as alpha helices, beta sheets, or functional polypeptide motifssuch as enzymatic active sites, helix-turn-helix motifs or other motifsknown to those skilled in the art. Once the database of features isopened at the state 306, the process 300 moves to a state 308 whereinthe first feature is read from the database. A comparison of theattribute of the first feature with the first sequence is then made at astate 310. A determination is then made at a decision state 316 whetherthe attribute of the feature was found in the first sequence. If theattribute was found, then the process 300 moves to a state 318 whereinthe name of the found feature is displayed to the user. The process 300then moves to a decision state 320 wherein a determination is madewhether move features exist in the database. If no more features doexist, then the process 300 terminates at an end state 324. However, ifmore features do exist in the database, then the process 300 reads thenext sequence feature at a state 326 and loops back to the state 310wherein the attribute of the next feature is compared against the firstsequence. If the feature attribute is not found in the first sequence atthe decision state 316, the process 300 moves directly to the decisionstate 320 in order to determine if any more features exist in thedatabase. Thus, in one aspect, the invention provides a computer programthat identifies open reading frames (ORFs).

A polypeptide or nucleic acid sequence of the invention may be storedand manipulated in a variety of data processor programs in a variety offormats. For example, a sequence can be stored as text in a wordprocessing file, such as MicrosoftWORD or WORDPERFECT or as an ASCIIfile in a variety of database programs familiar to those of skill in theart, such as DB2, SYBASE, or ORACLE. In addition, many computer programsand databases may be used as sequence comparison algorithms,identifiers, or sources of reference nucleotide sequences or polypeptidesequences to be compared to a nucleic acid sequence of the invention.The programs and databases used to practice the invention include, butare not limited to: MacPattern (EMBL), DiscoveryBase (MolecularApplications Group), GeneMine (Molecular Applications Group), Look(Molecular Applications Group), MacLook (Molecular Applications Group),BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol.Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci.USA, 85: 2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci.6:237-245, 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE(Molecular Simulations Inc.), Cerius2.DBAccess (Molecular SimulationsInc.), HypoGen (Molecular Simulations Inc.), Insight II, (MolecularSimulations Inc.), Discover (Molecular Simulations Inc.), CHARMm(Molecular Simulations Inc.), Felix (Molecular Simulations Inc.),DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular SimulationsInc.), Homology (Molecular Simulations Inc.), Modeler (MolecularSimulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/ProteinDesign (Molecular Simulations Inc.), WebLab (Molecular SimulationsInc.), WebLab Diversity Explorer (Molecular Simulations Inc.), GeneExplorer (Molecular Simulations Inc.), SeqFold (Molecular SimulationsInc.), the MDL Available Chemicals Directory database, the MDL Drug DataReport data base, the Comprehensive Medicinal Chemistry database,Derwent's World Drug Index database, the BioByteMasterFile database, theGenbank database, and the Genseqn database. Many other programs and databases would be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices, and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites, and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated or recombinant nucleic acids thathybridize under stringent conditions to nucleic acid of the invention,e.g., an exemplary sequence of the invention, e.g., a sequence as setforth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, etc., and subsequences thereof, or a nucleic acidthat encodes a polypeptide of the invention. The stringent conditionscan be highly stringent conditions, medium stringent conditions, lowstringent conditions, including the high and reduced stringencyconditions described herein.

In alternative embodiments, nucleic acids of the invention as defined bytheir ability to hybridize under stringent conditions can be betweenabout five residues and the full length of nucleic acid of theinvention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50,55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues inlength. Nucleic acids shorter than full length are also included. Thesenucleic acids can be useful as, e.g., hybridization probes, labelingprobes, PCR oligonucleotide probes, iRNA, antisense or sequencesencoding antibody binding peptides (epitopes), motifs, active sites andthe like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C.

Alternatively, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprising conditions at 42°C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequenceblocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/mlsheared and denatured salmon sperm DNA). In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency conditions comprising 35% formamide at a reduced temperatureof 35° C.

Following hybridization, the filter may be washed with 6×SSC, 0.5% SDSat 50° C. These conditions are considered to be “moderate” conditionsabove 25% formamide and “low” conditions below 25% formamide. A specificexample of “moderate” hybridization conditions is when the abovehybridization is conducted at 30% formamide. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 10% formamide.

The temperature range corresponding to a particular level of stringencycan be further narrowed by calculating the purine to pyrimidine ratio ofthe nucleic acid of interest and adjusting the temperature accordingly.Nucleic acids of the invention are also defined by their ability tohybridize under high, medium, and low stringency conditions as set forthin Ausubel and Sambrook. Variations on the above ranges and conditionsare well known in the art. Hybridization conditions are discussedfurther, below.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na⁺ concentration of approximately1M. Following hybridization, the filter may be washed with 2×SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate” conditions above 50° C. and “low” conditions below 50°C. A specific example of “moderate” hybridization conditions is when theabove hybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

However, the selection of a hybridization format is not critical—it isthe stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope of theinvention. Wash conditions used to identify nucleic acids within thescope of the invention include, e.g.: a salt concentration of about 0.02molar at pH 7 and a temperature of at least about 50° C. or about 55° C.to about 60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C.for about 15 minutes; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 15 to about 20 minutes; or, the hybridization complex is washedtwice with a solution with a salt concentration of about 2×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedtwice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or,equivalent conditions. See Sambrook, Tijssen and Ausubel for adescription of SSC buffer and equivalent conditions.

These methods may be used to isolate nucleic acids of the invention.

Oligonucleotides Probes and Methods for Using them

The invention also provides nucleic acid probes for identifying nucleicacids encoding a polypeptide with a hydrolase activity, e.g., anesterase, acylase, lipase, phospholipase or protease activity. In oneaspect, the probe comprises at least 10 consecutive bases of a nucleicacid of the invention. Alternatively, a probe of the invention can be atleast about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 110, 120, 130, 150, 160, 170, 180, 190, 200 or more, orabout 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of asequence as set forth in a nucleic acid of the invention. The probesidentify a nucleic acid by binding and/or hybridization. The probes canbe used in arrays of the invention, see discussion below, including,e.g., capillary arrays. The probes of the invention can also be used toisolate other nucleic acids or polypeptides.

The probes of the invention can be used to determine whether abiological sample, such as a soil sample, contains an organism having anucleic acid sequence of the invention (e.g., a hydrolase-encodingnucleic acid) or an organism from which the nucleic acid was obtained.In such procedures, a biological sample potentially harboring theorganism from which the nucleic acid was isolated is obtained andnucleic acids are obtained from the sample. The nucleic acids arecontacted with the probe under conditions which permit the probe tospecifically hybridize to any complementary sequences present in thesample. Where necessary, conditions which permit the probe tospecifically hybridize to complementary sequences may be determined byplacing the probe in contact with complementary sequences from samplesknown to contain the complementary sequence, as well as controlsequences which do not contain the complementary sequence. Hybridizationconditions, such as the salt concentration of the hybridization buffer,the formamide concentration of the hybridization buffer, or thehybridization temperature, may be varied to identify conditions whichallow the probe to hybridize specifically to complementary nucleic acids(see discussion on specific hybridization conditions).

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product. Manymethods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures, and dot blots. Protocols for each of theseprocedures are provided in Ausubel and Sambrook.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). In one aspect, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook (seediscussion on amplification reactions). In such procedures, the nucleicacids in the sample are contacted with the probes, the amplificationreaction is performed, and any resulting amplification product isdetected. The amplification product may be detected by performing gelelectrophoresis on the reaction products and staining the gel with anintercalator such as ethidium bromide. Alternatively, one or more of theprobes may be labeled with a radioactive isotope and the presence of aradioactive amplification product may be detected by autoradiographyafter gel electrophoresis.

Probes derived from sequences near the 3′ or 5′ ends of a nucleic acidsequence of the invention can also be used in chromosome walkingprocedures to identify clones containing additional, e.g., genomicsequences. Such methods allow the isolation of genes which encodeadditional proteins of interest from the host organism.

In one aspect, nucleic acid sequences of the invention are used asprobes to identify and isolate related nucleic acids. In some aspects,the so-identified related nucleic acids may be cDNAs or genomic DNAsfrom organisms other than the one from which the nucleic acid of theinvention was first isolated. In such procedures, a nucleic acid sampleis contacted with the probe under conditions which permit the probe tospecifically hybridize to related sequences. Hybridization of the probeto nucleic acids from the related organism is then detected using any ofthe methods described above.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.Hybridization may be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO4, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's, and 0.5 mg/ml polyriboadenylic acid.Approximately 2×107 cpm (specific activity 4-9×108 cpm/ug) of 32Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature (RT) in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH7.8, 1 mM Na2EDTA) containing 0.5% SDS, followed by a 30 minute wash infresh 1×SET at Tm-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, Tm, is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the Tm for a particular probe. The melting temperature of the probemay be calculated using the following exemplary formulas. For probesbetween 14 and 70 nucleotides in length the melting temperature (Tm) iscalculated using the formula: Tm=81.5+16.6(log Na+1)+0.41(fractionG+C)−(600/N) where N is the length of the probe. If the hybridization iscarried out in a solution containing formamide, the melting temperaturemay be calculated using the equation: Tm=81.5+16.6(logNa+1)+0.41(fraction G+C)−(0.63% formamide)−(600/N) where N is the lengthof the probe. Prehybridization may be carried out in 6×SSC, 5×Denhardt'sreagent, 0.5% SDS, 100 μg denatured fragmented salmon sperm DNA or6×SSC, 5×Denhardt's reagent, 0.5% SDS, 100 μg denatured fragmentedsalmon sperm DNA, 50% formamide. Formulas for SSC and Denhardt's andother solutions are listed, e.g., in Sambrook.

In one aspect, hybridization is conducted by adding the detectable probeto the prehybridization solutions listed above. Where the probecomprises double stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the Tm. For shorterprobes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the Tm. In one aspect, hybridizations in6×SSC are conducted at approximately 68° C. In one aspect,hybridizations in 50% formamide containing solutions are conducted atapproximately 42° C. All of the foregoing hybridizations would beconsidered to be under conditions of high stringency.

In one aspect, following hybridization, the filter is washed to removeany non-specifically bound detectable probe. The stringency used to washthe filters can also be varied depending on the nature of the nucleicacids being hybridized, the length of the nucleic acids beinghybridized, the degree of complementarity, the nucleotide sequencecomposition (e.g., GC v. AT content), and the nucleic acid type (e.g.,RNA v. DNA). Examples of progressively higher stringency conditionwashes are as follows: 2×SSC, 0.1% SDS at room temperature for 15minutes (low stringency); 0.1×SSC, 0.5% SDS at room temperature for 30minutes to 1 hour (moderate stringency); 0.1×SSC, 0.5% SDS for 15 to 30minutes at between the hybridization temperature and 68° C. (highstringency); and 0.15M NaCl for 15 minutes at 72° C. (very highstringency). A final low stringency wash can be conducted in 0.1×SSC atroom temperature. The examples above are merely illustrative of one setof conditions that can be used to wash filters. One of skill in the artwould know that there are numerous recipes for different stringencywashes.

Nucleic acids which have hybridized to the probe can be identified byautoradiography or other conventional techniques. The above proceduremay be modified to identify nucleic acids having decreasing levels ofhomology to the probe sequence. For example, to obtain nucleic acids ofdecreasing homology to the detectable probe, less stringent conditionsmay be used. For example, the hybridization temperature may be decreasedin increments of 5° C. from 68° C. to 42° C. in a hybridization bufferhaving a Na+ concentration of approximately 1M. Following hybridization,the filter may be washed with 2×SSC, 0.5% SDS at the temperature ofhybridization. These conditions are considered to be “moderate”conditions above 50° C. and “low” conditions below 50° C. An example of“moderate” hybridization conditions is when the above hybridization isconducted at 55° C. An example of “low stringency” hybridizationconditions is when the above hybridization is conducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6× SSC, containing formamide at a temperature of 42° C. In this case,the concentration of formamide in the hybridization buffer may bereduced in 5% increments from 50% to 0% to identify clones havingdecreasing levels of homology to the probe. Following hybridization, thefilter may be washed with 6×SSC, 0.5% SDS at 50° C. These conditions areconsidered to be “moderate” conditions above 25% formamide and “low”conditions below 25% formamide. A specific example of “moderate”hybridization conditions is when the above hybridization is conducted at30% formamide. A specific example of “low stringency” hybridizationconditions is when the above hybridization is conducted at 10%formamide.

These probes and methods of the invention can be used to isolate, oridentify (e.g., using an array), nucleic acids having a sequence with atleast about 950%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequenceidentity to a nucleic acid sequence of the invention comprising at leastabout 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350,400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or moreconsecutive bases thereof, and the sequences complementary thereto.Homology may be measured using an alignment algorithm, as discussedherein. For example, the homologous polynucleotides may have a codingsequence which is a naturally occurring allelic variant of one of thecoding sequences described herein. Such allelic variants may have asubstitution, deletion or addition of one or more nucleotides whencompared to a nucleic acid of the invention.

Additionally, the probes and methods of the invention may be used toisolate, or identify (e.g., using an array), nucleic acids which encodepolypeptides having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity (homology) to a polypeptide of theinvention comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 or more consecutive amino acids thereof as determined usinga sequence alignment algorithm, e.g., such as the FASTA version 3.0t78algorithm with the default parameters, or a BLAST 2.2.2 program withexemplary settings as set forth herein.

Inhibiting Expression of Hydrolases

The invention further provides for nucleic acids complementary to (e.g.,antisense sequences to) the nucleic acid sequences of the invention,e.g., hydrolase-encoding sequences. Antisense sequences are capable ofinhibiting the transport, splicing or transcription ofhydrolase-encoding genes. The inhibition can be effected through thetargeting of genomic DNA or messenger RNA. The inhibition can beeffected using DNA, e.g., an inhibitory ribozyme, or an RNA, e.g., adouble-stranded iRNA, comprising a sequence of the invention. Thetranscription or function of targeted nucleic acid can be inhibited, forexample, by hybridization and/or cleavage. The invention provides a setof inhibitors comprising oligonucleotides capable of binding hydrolasegene and/or message, in either case preventing or inhibiting theproduction or function of hydrolase. The association can be throughsequence specific hybridization. Another useful class of inhibitorsincludes oligonucleotides which cause inactivation or cleavage ofhydrolase message. The oligonucleotide can have enzyme activity whichcauses such cleavage, such as ribozymes. The oligonucleotide can bechemically modified or conjugated to an enzyme or composition capable ofcleaving the complementary nucleic acid. One may screen a pool of manydifferent such oligonucleotides for those with the desired activity.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindinghydrolase message which can inhibit hydrolase activity by targeting mRNAor genomic DNA. Strategies for designing antisense oligonucleotides arewell described in the scientific and patent literature, and the skilledartisan can design such hydrolase oligonucleotides using the novelreagents of the invention. For example, gene walking/RNA mappingprotocols to screen for effective antisense oligonucleotides are wellknown in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183,describing an RNA mapping assay, which is based on standard moleculartechniques to provide an easy and reliable method for potent antisensesequence selection. See also Smith (2000) Eur. J. Pharm. Sci.11:191-198.

In one aspect, recombinantly generated, or, isolated naturally occurringnucleic acids are used as antisense oligonucleotides. The antisenseoligonucleotides can be of any length; for example, in alternativeaspects, the antisense oligonucleotides are between about 5 to 100,about 10 to 80, about 15 to 60, about 18 to 40. The antisenseoligonucleotides can be single stranded or double-stranded RNA or DNA.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl) glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N. J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisensehydrolase sequences of the invention (see, e.g., Gold (1995) J. of Biol.Chem. 270:13581-13584).

Inhibitory Ribozymes

The invention provides for with ribozymes capable of binding hydrolasemessage that can inhibit hydrolase activity by targeting mRNA.Strategies for designing ribozymes and selecting the hydrolase-specificantisense sequence for targeting are well described in the scientificand patent literature, and the skilled artisan can design such ribozymesusing the novel reagents of the invention. Ribozymes act by binding to atarget RNA through the target RNA binding portion of a ribozyme which isheld in close proximity to an enzymatic portion of the RNA that cleavesthe target RNA. Thus, the ribozyme recognizes and binds a target RNAthrough complementary basepairing, and once bound to the correct site,acts enzymatically to cleave and inactivate the target RNA. Cleavage ofa target RNA in such a manner will destroy its ability to directsynthesis of an encoded protein if the cleavage occurs in the codingsequence. After a ribozyme has bound and cleaved its RNA target, it istypically released from that RNA and so can bind and cleave new targetsrepeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The enzymatic ribozyme RNA molecule can be formed in a hammerhead motif,but may also be formed in the motif of a hairpin, hepatitis delta virus,group I intron or RnaseP-like RNA (in association with an RNA guidesequence). Examples of such hammerhead motifs are described by Rossi(1992) Aids Research and Human Retroviruses 8:183; hairpin motifs byHampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.18:299; the hepatitis delta virus motif by Perrotta (1992) Biochemistry31:16; the RNaseP motif by Guerrier-Takada (1983) Cell 35:849; and thegroup I intron by Cech U.S. Pat. No. 4,987,071. The recitation of thesespecific motifs is not intended to be limiting; those skilled in the artwill recognize that an enzymatic RNA molecule of this invention has aspecific substrate binding site complementary to one or more of thetarget gene RNA regions, and has nucleotide sequence within orsurrounding that substrate binding site which imparts an RNA cleavingactivity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising a hydrolase sequence of theinvention. The RNAi molecule comprises a double-stranded RNA (dsRNA)molecule. The RNAi can inhibit expression of a hydrolase gene. In oneaspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length. While the invention is not limited byany particular mechanism of action, the RNAi can enter a cell and causethe degradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed todouble-stranded RNA (dsRNA), mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi). Apossible basic mechanism behind RNAi is the breaking of adouble-stranded RNA (dsRNA) matching a specific gene sequence into shortpieces called short interfering RNA, which trigger the degradation ofmRNA that matches its sequence. In one aspect, the RNAi's of theinvention are used in gene-silencing therapeutics, see, e.g., Shuey(2002) Drug Discov. Today 7:1040-1046. In one aspect, the inventionprovides methods to selectively degrade RNA using the RNAi's of theinvention. The process may be practiced in vitro, ex vivo or in vivo. Inone aspect, the RNAi molecules of the invention can be used to generatea loss-of-function mutation in a cell, an organ or an animal. Methodsfor making and using RNAi molecules for selectively degrade RNA are wellknown in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824;6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding a hydrolase or an antibodyof the invention. These methods can be repeated or used in variouscombinations to generate hydrolases or antibodies having an altered ordifferent activity or an altered or different stability from that of ahydrolase or antibody encoded by the template nucleic acid. Thesemethods also can be repeated or used in various combinations, e.g., togenerate variations in gene/message expression, message translation ormessage stability. In another aspect, the genetic composition of a cellis altered by, e.g., modification of a homologous gene ex vivo, followedby its reinsertion into the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods, see, e.g., U.S. Pat. No. 6,361,974. Methods forrandom mutation of genes are well known in the art, see, e.g., U.S. Pat.No. 5,830,696. For example, mutagens can be used to randomly mutate agene. Mutagens include, e.g., ultraviolet light or gamma irradiation, ora chemical mutagen, e.g., mitomycin, nitrous acid, photoactivatedpsoralens, alone or in combination, to induce DNA breaks amenable torepair by recombination. Other chemical mutagens include, for example,sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.Other mutagens are analogues of nucleotide precursors, e.g.,nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or acombination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye (1986) “Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols used in the methods of the invention include pointmismatch repair (Kramer (1984) “Point Mismatch Repair” Cell 38:879-887),mutagenesis using repair-deficient host strains (Carter et al. (1985)“Improved oligonucleotide site-directed mutagenesis using M13 vectors”Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Additional protocols used in the methods of the invention include thosediscussed in U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997),“Methods for In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmeret al. (Sep. 22, 1998) “Methods for Generating Polynucleotides havingDesired Characteristics by Iterative Selection and Recombination;” U.S.Pat. No. 5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis byRandom Fragmentation and Reassembly;” U.S. Pat. No. 5,834,252 toStemmer, et al. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;”U.S. Pat. No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methodsand Compositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Protocols that can be used to practice the invention (providing detailsregarding various diversity generating methods) are described, e.g., inU.S. patent application Ser. No. (USSN) 09/407,800, “SHUFFLING OF CODONALTERED GENES” by Patten et al. filed Sep. 28, 1999; “EVOLUTION OF WHOLECELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” by del Cardayreet al., U.S. Pat. No. 6,379,964; “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACIDRECOMBINATION” by Crameri et al., U.S. Pat. Nos. 6,319,714; 6,368,861;6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; “USE OF CODON-VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., U.S.Pat. No. 6,436,675; “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, e.g.“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000(U.S. Ser. No. 09/618,579); “METHODS OF POPULATING DATA STRUCTURES FORUSE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan.18, 2000 (PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and U.S. Pat.Nos. 6,177,263; 6,153,410.

Non-stochastic, or “directed evolution,” methods include, e.g.,saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or acombination thereof are used to modify the nucleic acids of theinvention to generate hydrolases with new or altered properties (e.g.,activity under highly acidic or alkaline conditions, high temperatures,and the like). Polypeptides encoded by the modified nucleic acids can bescreened for an activity before testing for proteolytic or otheractivity. Any testing modality or protocol can be used, e.g., using acapillary array platform. See, e.g., U.S. Pat. Nos. 6,361,974;6,280,926; 5,939,250.

Saturation Mutagenesis, or, GSSM

In one aspect of the invention, non-stochastic gene modification, a“directed evolution process,” is used to generate hydrolases andantibodies with new or altered properties. Variations of this methodhave been termed “gene site-saturation mutagenesis,” “site-saturationmutagenesis,” “saturation mutagenesis” or simply “GSSM.” It can be usedin combination with other mutagenization processes. See, e.g., U.S. Pat.Nos. 6,171,820; 6,238,884. In one aspect, GSSM comprises providing atemplate polynucleotide and a plurality of oligonucleotides, whereineach oligonucleotide comprises a sequence homologous to the templatepolynucleotide, thereby targeting a specific sequence of the templatepolynucleotide, and a sequence that is a variant of the homologous gene;generating progeny polynucleotides comprising non-stochastic sequencevariations by replicating the template polynucleotide with theoligonucleotides, thereby generating polynucleotides comprisinghomologous gene sequence variations.

In one aspect, codon primers containing a degenerate N,N,G/T sequenceare used to introduce point mutations into a polynucleotide, so as togenerate a set of progeny polypeptides in which a full range of singleamino acid substitutions is represented at each amino acid position,e.g., an amino acid residue in an enzyme active site or ligand bindingsite targeted to be modified. These oligonucleotides can comprise acontiguous first homologous sequence, a degenerate N,N,G/T sequence,and, optionally, a second homologous sequence. The downstream progenytranslational products from the use of such oligonucleotides include allpossible amino acid changes at each amino acid site along thepolypeptide, because the degeneracy of the N,N,G/T sequence includescodons for all 20 amino acids. In one aspect, one such degenerateoligonucleotide (comprised of, e.g., one degenerate N,N,G/T cassette) isused for subjecting each original codon in a parental polynucleotidetemplate to a full range of codon substitutions. In another aspect, atleast two degenerate cassettes are used—either in the sameoligonucleotide or not, for subjecting at least two original codons in aparental polynucleotide template to a full range of codon substitutions.For example, more than one N,N,G/T sequence can be contained in oneoligonucleotide to introduce amino acid mutations at more than one site.This plurality of N,N,G/T sequences can be directly contiguous, orseparated by one or more additional nucleotide sequence(s). In anotheraspect, oligonucleotides serviceable for introducing additions anddeletions can be used either alone or in combination with the codonscontaining an N,N,G/T sequence, to introduce any combination orpermutation of amino acid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequence comprisedof only one N, where said N can be in the first second or third positionof the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position×100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can optionally be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,hydrolases, e.g., esterases, acylases, lipases, phospholipases orproteases) molecules such that all 20 natural amino acids arerepresented at the one specific amino acid position corresponding to thecodon position mutagenized in the parental polynucleotide (other aspectsuse less than all 20 natural combinations). The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g. cloned into asuitable host, e.g., E. coli host, using, e.g., an expression vector)and subjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide, such as increasedproteolytic activity under alkaline or acidic conditions), it can besequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined—6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In another aspect, site-saturation mutagenesis can be used together withanother stochastic or non-stochastic means to vary sequence, e.g.,synthetic ligation reassembly (see below), shuffling, chimerization,recombination and other mutagenizing processes and mutagenizing agents.This invention provides for the use of any mutagenizing process(es),including saturation mutagenesis, in an iterative manner.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate hydrolases and antibodies with new or alteredproperties. SLR is a method of ligating oligonucleotide fragmentstogether non-stochastically. This method differs from stochasticoligonucleotide shuffling in that the nucleic acid building blocks arenot shuffled, concatenated or chimerized randomly, but rather areassembled non-stochastically. See, e.g., U.S. patent application Ser.No. (USSN) 09/332,835 entitled “Synthetic Ligation Reassembly inDirected Evolution” and filed on Jun. 14, 1999 (“U.S. Ser. No.09/332,835”). In one aspect, SLR comprises the following steps: (a)providing a template polynucleotide, wherein the template polynucleotidecomprises sequence encoding a homologous gene; (b) providing a pluralityof building block polynucleotides, wherein the building blockpolynucleotides are designed to cross-over reassemble with the templatepolynucleotide at a predetermined sequence, and a building blockpolynucleotide comprises a sequence that is a variant of the homologousgene and a sequence homologous to the template polynucleotide flankingthe variant sequence; (c) combining a building block polynucleotide witha template polynucleotide such that the building block polynucleotidecross-over reassembles with the template polynucleotide to generatepolynucleotides comprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10100 different chimeras. SLR can be used to generatelibraries comprised of over 101000 different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled. In one aspect of this method, the sequences of aplurality of parental nucleic acid templates are aligned in order toselect one or more demarcation points. The demarcation points can belocated at an area of homology, and are comprised of one or morenucleotides. These demarcation points are preferably shared by at leasttwo of the progenitor templates. The demarcation points can thereby beused to delineate the boundaries of oligonucleotide building blocks tobe generated in order to rearrange the parental polynucleotides. Thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the finalchimeric progeny molecules. A demarcation point can be an area ofhomology (comprised of at least one homologous nucleotide base) sharedby at least two parental polynucleotide sequences. Alternatively, ademarcation point can be an area of homology that is shared by at leasthalf of the parental polynucleotide sequences, or, it can be an area ofhomology that is shared by at least two thirds of the parentalpolynucleotide sequences. Even more preferably a serviceable demarcationpoints is an area of homology that is shared by at least three fourthsof the parental polynucleotide sequences, or, it can be shared by atalmost all of the parental polynucleotide sequences. In one aspect, ademarcation point is an area of homology that is shared by all of theparental polynucleotide sequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in another aspect,the assembly order (i.e. the order of assembly of each building block inthe 5′ to 3 sequence of each finalized chimeric nucleic acid) in eachcombination is by design (or non-stochastic) as described above. Becauseof the non-stochastic nature of this invention, the possibility ofunwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedpreferably comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecular homologous demarcationpoints and thus to allow an increased number of couplings to be achievedamong the building blocks, which in turn allows a greater number ofprogeny chimeric molecules to be generated.

In another aspect, the synthetic nature of the step in which thebuilding blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g. by mutagenesis) or in an in vivoprocess (e.g. by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

In one aspect, a nucleic acid building block is used to introduce anintron. Thus, functional introns are introduced into a man-made genemanufactured according to the methods described herein. The artificiallyintroduced intron(s) can be functional in a host cells for gene splicingmuch in the way that naturally-occurring introns serve functionally ingene splicing.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate hydrolases andantibodies with new or altered properties. Optimized directed evolutionis directed to the use of repeated cycles of reductive reassortment,recombination and selection that allow for the directed molecularevolution of nucleic acids through recombination. Optimized directedevolution allows generation of a large population of evolved chimericsequences, wherein the generated population is significantly enrichedfor sequences that have a predetermined number of crossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 10¹³ chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide preferably includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Additional information can also be found, e.g., in U.S.Ser. No. 09/332,835; U.S. Pat. No. 6,361,974.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding a polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

Determining Crossover Events

Aspects of the invention include a system and software that receive adesired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is preferably performed in MATLAB™ (The Mathworks, Natick, Mass.)a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example a nucleic acid (or, the nucleic acid) responsiblefor an altered hydrolase or antibody phenotype is identified,re-isolated, again modified, re-tested for activity. This process can beiteratively repeated until a desired phenotype is engineered. Forexample, an entire biochemical anabolic or catabolic pathway can beengineered into a cell, including proteolytic activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new hydrolase phenotype), itcan be removed as a variable by synthesizing larger parentaloligonucleotides that include the sequence to be removed. Sinceincorporating the sequence within a larger sequence prevents anycrossover events, there will no longer be any variation of this sequencein the progeny polynucleotides. This iterative practice of determiningwhich oligonucleotides are most related to the desired trait, and whichare unrelated, allows more efficient exploration all of the possibleprotein variants that might be provide a particular trait or activity.

In Vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention thatprovide variants of polypeptides of the invention, e.g., antibodies,hydrolases, and the like. In vivo shuffling can be performed utilizingthe natural property of cells to recombine multimers. Whilerecombination in vivo has provided the major natural route to moleculardiversity, genetic recombination remains a relatively complex processthat involves 1) the recognition of homologies; 2) strand cleavage,strand invasion, and metabolic steps leading to the production ofrecombinant chiasma; and finally 3) the resolution of chiasma intodiscrete recombined molecules. The formation of the chiasma requires therecognition of homologous sequences.

In one aspect, the invention provides a method for producing a hybridpolynucleotide from at least a first polynucleotide and a secondpolynucleotide. The invention can be used to produce a hybridpolynucleotide by introducing at least a first polynucleotide and asecond polynucleotide which share at least one region of partialsequence homology into a suitable host cell. The regions of partialsequence homology promote processes which result in sequencereorganization producing a hybrid polynucleotide. The term “hybridpolynucleotide”, as used herein, is any nucleotide sequence whichresults from the method of the present invention and contains sequencefrom at least two original polynucleotide sequences. Such hybridpolynucleotides can result from intermolecular recombination eventswhich promote sequence integration between DNA molecules. In addition,such hybrid polynucleotides can result from intramolecular reductivereassortment processes which utilize repeated sequences to alter anucleotide sequence within a DNA molecule.

Producing Sequence Variants

The invention also provides methods of making sequence variants of thenucleic acid and hydrolase and antibody sequences of the invention orisolating hydrolases using the nucleic acids and polypeptides of theinvention. In one aspect, the invention provides for variants of ahydrolase gene of the invention, which can be altered by any means,including, e.g., random or stochastic methods, or, non-stochastic, or“directed evolution,” methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. These nucleotidedifferences can result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989)and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl₂, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids isevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/:l in a solution of 0.2 mM of each dNTP, 2.2mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In some aspects,random mutations in a sequence of interest are generated by propagatingthe sequence of interest in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described, e.g., inPCT Publication No. WO 91/16427.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815.

In some aspects, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described,e.g., in Delegrave (1993) Biotechnology Res. 11:1548-1552. Random andsite-directed mutagenesis are described, e.g., in Arnold (1993) CurrentOpinion in Biotechnology 4:450-455.

In some aspects, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in, e.g., U.S.Pat. Nos. 5,965,408; 5,939,250.

The invention also provides variants of polypeptides of the inventioncomprising sequences in which one or more of the amino acid residues(e.g., of an exemplary polypeptide, such as SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, etc.) are substituted with a conserved ornon-conserved amino acid residue (e.g., a conserved amino acid residue)and such substituted amino acid residue may or may not be one encoded bythe genetic code. Conservative substitutions are those that substitute agiven amino acid in a polypeptide by another amino acid of likecharacteristics. Thus, polypeptides of the invention include those withconservative substitutions of sequences of the invention, e.g., theexemplary sequences of the invention, such as SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, etc., including but not limited to thefollowing replacements: replacements of an aliphatic amino acid such asAlanine, Valine, Leucine and Isoleucine with another aliphatic aminoacid; replacement of a Serine with a Threonine or vice versa;replacement of an acidic residue such as Aspartic acid and Glutamic acidwith another acidic residue; replacement of a residue bearing an amidegroup, such as Asparagine and Glutamine, with another residue bearing anamide group; exchange of a basic residue such as Lysine and Argininewith another basic residue; and replacement of an aromatic residue suchas Phenylalanine, Tyrosine with another aromatic residue. Other variantsare those in which one or more of the amino acid residues of thepolypeptides of the invention includes a substituent group.

Other variants within the scope of the invention are those in which thepolypeptide is associated with another compound, such as a compound toincrease the half-life of the polypeptide, for example, polyethyleneglycol. Additional variants within the scope of the invention are thosein which additional amino acids are fused to the polypeptide, such as aleader sequence, a secretory sequence, a proprotein sequence or asequence which facilitates purification, enrichment, or stabilization ofthe polypeptide. In some aspects, the variants, fragments, derivativesand analogs of the polypeptides of the invention retain the samebiological function or activity as the exemplary polypeptides, e.g., aproteolytic activity, as described herein. In other aspects, thevariant, fragment, derivative, or analog includes a proprotein, suchthat the variant, fragment, derivative, or analog can be activated bycleavage of the proprotein portion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying hydrolase-encoding nucleicacids to modify codon usage. In one aspect, the invention providesmethods for modifying codons in a nucleic acid encoding a hydrolase toincrease or decrease its expression in a host cell, e.g., a bacterial,insect, mammalian, yeast or plant cell. The invention also providesnucleic acids encoding a hydrolase modified to increase its expressionin a host cell, hydrolase so modified, and methods of making themodified hydrolases. The method comprises identifying a “non-preferred”or a “less preferred” codon in hydrolase-encoding nucleic acid andreplacing one or more of these non-preferred or less preferred codonswith a “preferred codon” encoding the same amino acid as the replacedcodon and at least one non-preferred or less preferred codon in thenucleic acid has been replaced by a preferred codon encoding the sameamino acid. A preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli; gram positive bacteria, such as any Bacillus (e.g., B. cereus orB. subtilis) or Streptomyces, Lactobacillus gasseri, Lactococcus lactis,Lactococcus cremoris. Exemplary host cells also include eukaryoticorganisms, e.g., various yeast, such as Saccharomyces sp., includingSaccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris,and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, andmammalian cells and cell lines and insect cells and cell lines. Thus,the invention also includes nucleic acids and polypeptides optimized forexpression in these organisms and species.

For example, the codons of a nucleic acid encoding a hydrolase isolatedfrom a bacterial cell are modified such that the nucleic acid isoptimally expressed in a bacterial cell different from the bacteria fromwhich the hydrolase was derived, a yeast, a fungi, a plant cell, aninsect cell or a mammalian cell. Methods for optimizing codons are wellknown in the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int.J. Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188;Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect.Immun. 69:7250-7253, describing optimizing codons in mouse systems;Outchkourov (2002) Protein Expr. Purif. 24:18-24, describing optimizingcodons in yeast; Feng (2000) Biochemistry 39:15399-15409, describingoptimizing codons in E. coli; Humphreys (2000) Protein Expr. Purif.20:252-264, describing optimizing codon usage that affects secretion inE. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide (e.g., a hydrolase or an antibody of the invention),an expression cassette, a vector, a transfected or a transformed cell ofthe invention. The transgenic non-human animals can be, e.g., goats,rabbits, sheep, pigs, cows, rats and mice, comprising the nucleic acidsof the invention. These animals can be used, e.g., as in vivo models tostudy hydrolase activity, or, as models to screen for agents that changethe hydrolase activity in vivo. The coding sequences for thepolypeptides to be expressed in the transgenic non-human animals can bedesigned to be constitutive, or, under the control of tissue-specific,developmental-specific or inducible transcriptional regulatory factors.Transgenic non-human animals can be designed and generated using anymethod known in the art; see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992;6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854;5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742;5,087,571, describing making and using transformed cells and eggs andtransgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g.,Pollock (1999) J. Immunol. Methods 231:147-157, describing theproduction of recombinant proteins in the milk of transgenic dairyanimals; Baguisi (1999) Nat. Biotechnol. 17:456-461, demonstrating theproduction of transgenic goats. U.S. Pat. No. 6,211,428, describesmaking and using transgenic non-human mammals which express in theirbrains a nucleic acid construct comprising a DNA sequence. U.S. Pat. No.5,387,742, describes injecting cloned recombinant or synthetic DNAsequences into fertilized mouse eggs, implanting the injected eggs inpseudo-pregnant females, and growing to term transgenic mice whose cellsexpress proteins related to the pathology of Alzheimer's disease. U.S.Pat. No. 6,187,992, describes making and using a transgenic mouse whosegenome comprises a disruption of the gene encoding amyloid precursorprotein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express an endogenous gene, which is replacedwith a gene expressing a hydrolase of the invention, or, a fusionprotein comprising a hydrolase of the invention. As noted above,functional knockouts can also be generated using antisense sequences ofthe invention, e.g., double-stranded RNAi molecules.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (e.g., a hydrolase or an antibody of the invention),an expression cassette or vector or a transfected or transformed cell ofthe invention. The transgenic plant can be dicotyledonous (a dicot) ormonocotyledonous (a monocot). The invention also provides methods ofmaking and using these transgenic plants and seeds. The transgenic plantor plant cell expressing a polypeptide of the present invention may beconstructed in accordance with any method known in the art. See, forexample, U.S. Pat. No. 6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. For example, nucleic acids orexpression constructs can be introduced into the genome of a desiredplant host, or, the nucleic acids or expression constructs can beepisomes. Introduction into the genome of a desired plant can be suchthat the host's hydrolase production is regulated by endogenoustranscriptional or translational control elements. The invention alsoprovides “knockout plants” where insertion of gene sequence by, e.g.,homologous recombination, has disrupted the expression of the endogenousgene. Means to generate “knockout” plants are well-known in the art,see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao(1995) Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant, e.g., on oilseed producing plants, includingrice bran, rapeseed (canola), sunflower, olive, palm or soy, and thelike, or on glucose or starch-producing plants, such as corn, potato,wheat, rice, barley, and the like. Nucleic acids of the invention can beused to manipulate metabolic pathways of a plant in order to optimize oralter host's expression of a hydrolase or a substrate or product of ahydrolase, e.g., an oil, a lipid, such as a mono-, di- ortri-acylglyceride and the like. The can change the ratios of lipids,lipid conversion and turnover in a plant. This can facilitate industrialprocessing of a plant. Alternatively, hydrolases of the invention can beused in production of a transgenic plant to produce a compound notnaturally produced by that plant. This can lower production costs orcreate a novel product.

In one aspect, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence of the invention islikely to have a higher percentage of A-T nucleotide pairs compared tothat seen in a plant, some of which prefer G-C nucleotide pairs.Therefore, A-T nucleotides in the coding sequence can be substitutedwith G-C nucleotides without significantly changing the amino acidsequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, optionally, marker genes into a targetexpression construct (e.g., a plasmid, a phage), along with positioningof the promoter and the terminator sequences. This can involvetransferring the modified gene into the plant through a suitable method.For example, a construct may be introduced directly into the genomic DNAof the plant cell using techniques such as electroporation andmicroinjection of plant cell protoplasts, or the constructs can beintroduced directly to plant tissue using ballistic methods, such as DNAparticle bombardment. For example, see, e.g., Christou (1997) Plant Mol.Biol. 35:197-203; Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein(1987) Nature 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69,discussing use of particle bombardment to introduce transgenes intowheat; and Adam (1997) supra, for use of particle bombardment tointroduce YACs into plant cells. For example, Rinehart (1997) supra,used particle bombardment to generate transgenic cotton plants.Apparatus for accelerating particles is described U.S. Pat. No.5,015,580; and, the commercially available BioRad (Biolistics) PDS-2000particle acceleration instrument; see also, John, U.S. Pat. No.5,608,148; and Ellis, U.S. Pat. No. 5,681,730, describingparticle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with anucleic acids, e.g., an expression construct. Although plantregeneration from protoplasts is not easy with cereals, plantregeneration is possible in legumes using somatic embryogenesis fromprotoplast derived callus. Organized tissues can be transformed withnaked DNA using gene gun technique, where DNA is coated on tungstenmicroprojectiles, shot 1/100th the size of cells, which carry the DNAdeep into cells and organelles. Transformed tissue is then induced toregenerate, usually by somatic embryogenesis. This technique has beensuccessful in several cereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci USA 80:4803;Thykjaer (1997) supra; Park (1996) Plant Mol. Biol. 32:1135-1148,discussing T-DNA integration into genomic DNA. See also D'Halluin, U.S.Pat. No. 5,712,135, describing a process for the stable integration of aDNA comprising a gene that is functional in a cell of a cereal, or othermonocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. Since transgenic expression of the nucleicacids of the invention leads to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a plant of the invention and anotherplant. The desired effects (e.g., expression of the polypeptides of theinvention to produce a plant with altered, increased and/or decreasedlipid or oil content) can be enhanced when both parental plants expressthe polypeptides of the invention. The desired effects can be passed tofuture plant generations by standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in orinserted in any plant or seed. Transgenic plants of the invention can bedicotyledonous or monocotyledonous. Examples of monocot transgenicplants of the invention are grasses, such as meadow grass (blue grass,Poa), forage grass such as festuca, lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn). Examples of dicot transgenic plants of the inventionare tobacco, legumes, such as lupins, potato, sugar beet, pea, bean andsoybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants which contain fiber cells, including, e.g., cotton,silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush,winterfat, balsa, ramie, kenaf, hemp, rosette, jute, sisal abaca andflax. In alternative embodiments, the transgenic plants of the inventioncan be members of the genus Gossypium, including members of anyGossypium species, such as G. arboreum; G. herbaceum, G. barbadense, andG. hirsutum.

The invention also provides for transgenic plants to be used forproducing large amounts of the polypeptides (e.g., antibodies,hydrolases) of the invention. For example, see Palmgren (1997) TrendsGenet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producing humanmilk protein beta-casein in transgenic potato plants using anauxin-inducible, bidirectional mannopine synthase (mas1′,2′) promoterwith Agrobacterium tumefaciens-mediated leaf disc transformationmethods).

Using known procedures, one of skill can screen for plants of theinvention by detecting the increase or decrease of transgene mRNA orprotein in transgenic plants. Means for detecting and quantitation ofmRNAs or proteins are well known in the art.

In one aspect, the invention produces fatty acids or fatty acidderivatives from transgenic plants of the invention, e.g., transgenicoleaginous plants. In one aspect, transgenic oleaginous plantscomprising at least one hydrolase of the invention are produced. In oneaspect, the transgenic plant comprises a hydrolase gene operably linkedto a promoter, permitting an expression of the gene either in cellular,extracellular or tissue compartments other than those in which the plantlipids accumulate, or permitting exogenous induction of the hydrolase.In one aspect, seeds and/or fruits containing the lipids of the plantsare collected, the seeds and/or fruits are crushed (if necessary afterhydrolase (e.g., lipase) gene-induction treatment) so as to bring intocontact the lipids and hydrolase of the invention contained in the seedsand/or fruits. The mixture can be allowed to incubate to allow enzymatichydrolysis of the lipids of the ground material by catalytic action ofthe lipase of the invention contained in the crushed material. In oneaspect, the fatty acids formed by the hydrolysis are extracted and/orare converted in order to obtain the desired fatty acid derivatives.

This enzymatic hydrolysis process of the invention uses mild operatingconditions and can be small-scale and use inexpensive installations. Inthis aspect the plant of the invention is induced to produce thehydrolase for transformation of plant lipids. Using this strategy, theenzyme is prevented from coming into contact with stored plant lipids soas to avoid any risk of premature hydrolysis (“self-degradation of theplant”) before harvesting. The crushing and incubating units can belight and small-scale; many are known in the agricultural industry andcan be carried out at the sites where the plants are harvested.

In one aspect, transgenic plants of the invention are produced bytransformation of natural oleaginous plants. The genetically transformedplants of the invention are then reproduced sexually so as to producetransgenic seeds of the invention. These seeds can be used to obtaintransgenic plant progeny.

In one aspect, the hydrolase gene is operably linked to an induciblepromoter to prevent any premature contact of hydrolase and plant lipid.This promoter can direct the expression of the gene in compartmentsother than those where the lipids accumulate or the promoter caninitiate the expression of the hydrolase at a desired time by anexogenous induction.

Polypeptides and Peptides

The invention provides isolated or recombinant polypeptides having asequence identity (e.g., at least 50% sequence identity) to an exemplarysequence of the invention, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ IDNO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ IDNO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116,SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ IDNO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143,SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ IDNO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172,SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ IDNO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200,SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ IDNO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228,SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ IDNO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256,SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ IDNO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284,SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ IDNO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312,SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ IDNO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340,SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ IDNO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368,SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ IDNO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ ID NO:384, SEQ ID NO:386, SEQID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, SEQ ID NO:396,SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402, SEQ ID NO:404, SEQ IDNO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ ID NO:414, SEQID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQ ID NO:422, SEQ ID NO:424,SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, SEQ ID NO:432, SEQ IDNO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:442, SEQID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ ID NO:450, SEQ ID NO:452,SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458, SEQ ID NO:460, SEQ IDNO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ ID NO:470, SEQID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQ ID NO:478, SEQ ID NO:480,SEQ ID NO:482, SEQ ID NO:484, SEQ ID NO:486, SEQ ID NO:488, SEQ IDNO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:496, SEQ ID NO:498, SEQID NO:500, SEQ ID NO:502, SEQ ID NO:504, SEQ ID NO:506, SEQ ID NO:508,SEQ ID NO:510, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:516, SEQ IDNO:518, SEQ ID NO:520, SEQ ID NO:522, SEQ ID NO:524, SEQ ID NO:526, SEQID NO:528, SEQ ID NO:530, SEQ ID NO:532, SEQ ID NO:534, SEQ ID NO:536,SEQ ID NO:538, SEQ ID NO:540, SEQ ID NO:542, SEQ ID NO:544, SEQ IDNO:546, SEQ ID NO:548, SEQ ID NO:550, SEQ ID NO:552, SEQ ID NO:554, SEQID NO:556, SEQ ID NO:558, SEQ ID NO:560, SEQ ID NO:562, SEQ ID NO:564,SEQ ID NO:566, SEQ ID NO:568, SEQ ID NO:570, SEQ ID NO:572, SEQ IDNO:574, SEQ ID NO:576, SEQ ID NO:578, SEQ ID NO:580, SEQ ID NO:582, SEQID NO:584, SEQ ID NO:586, SEQ ID NO:588, SEQ ID NO:590, SEQ ID NO:592,SEQ ID NO:594, SEQ ID NO:596, SEQ ID NO:598, SEQ ID NO:600, SEQ IDNO:602, SEQ ID NO:604, SEQ ID NO:606, SEQ ID NO:608, SEQ ID NO:610, SEQID NO:612, SEQ ID NO:614, SEQ ID NO:616, SEQ ID NO:618, SEQ ID NO:620,SEQ ID NO:622, SEQ ID NO:624, SEQ ID NO:626, SEQ ID NO:628, SEQ IDNO:630, SEQ ID NO:632, SEQ ID NO:634, SEQ ID NO:636, SEQ ID NO:638, SEQID NO:640, SEQ ID NO:642, SEQ ID NO:644, SEQ ID NO:646, SEQ ID NO:648,SEQ ID NO:650, SEQ ID NO:652, SEQ ID NO:654, SEQ ID NO:656, SEQ IDNO:658, SEQ ID NO:660, SEQ ID NO:662, SEQ ID NO:664, SEQ ID NO:666, SEQID NO:668, SEQ ID NO:670, SEQ ID NO:672, SEQ ID NO:674, SEQ ID NO:676,SEQ ID NO:678, SEQ ID NO:680, SEQ ID NO:682, SEQ ID NO:684, SEQ IDNO:686, SEQ ID NO:688, SEQ ID NO:690, SEQ ID NO:692, SEQ ID NO:694, SEQID NO:696, SEQ ID NO:698, SEQ ID NO:700, SEQ ID NO:702, SEQ ID NO:704,SEQ ID NO:706, SEQ ID NO:708, SEQ ID NO:710, SEQ ID NO:712, SEQ IDNO:714, SEQ ID NO:716, SEQ ID NO:718, SEQ ID NO:720, SEQ ID NO:722, SEQID NO:724, SEQ ID NO:726, SEQ ID NO:728, SEQ ID NO:730, SEQ ID NO:732,SEQ ID NO:734, SEQ ID NO:736, SEQ ID NO:738, SEQ ID NO:740, SEQ IDNO:742, SEQ ID NO:744, SEQ ID NO:746, SEQ ID NO:748, SEQ ID NO:750, SEQID NO:752, SEQ ID NO:754, SEQ ID NO:756, SEQ ID NO:758, SEQ ID NO:760,SEQ ID NO:762, SEQ ID NO:764, SEQ ID NO:766, SEQ ID NO:768, SEQ IDNO:770, SEQ ID NO:772, SEQ ID NO:774, SEQ ID NO:776, SEQ ID NO:778, SEQID NO:780, SEQ ID NO:782, SEQ ID NO:784, SEQ ID NO:786, SEQ ID NO:788,SEQ ID NO:790, SEQ ID NO:792, SEQ ID NO:794, SEQ ID NO:796, SEQ IDNO:798, SEQ ID NO:800, SEQ ID NO:802, SEQ ID NO:804, SEQ ID NO:808, SEQID NO:808, SEQ ID NO:810, SEQ ID NO:812, SEQ ID NO:814, SEQ ID NO:816,SEQ ID NO:818, SEQ ID NO:820, SEQ ID NO:822, SEQ ID NO:824, SEQ IDNO:826, SEQ ID NO:828, SEQ ID NO:830, SEQ ID NO:832, SEQ ID NO:834, SEQID NO:836, SEQ ID NO:838, SEQ ID NO:840, SEQ ID NO:842, SEQ ID NO:844,SEQ ID NO:846, SEQ ID NO:848, SEQ ID NO:850, SEQ ID NO:852, SEQ IDNO:854, SEQ ID NO:856, SEQ ID NO:858, SEQ ID NO:860, SEQ ID NO:862, SEQID NO:864, SEQ ID NO:866, SEQ ID NO:868, SEQ ID NO:870, SEQ ID NO:872,SEQ ID NO:874, SEQ ID NO:876, SEQ ID NO:878, SEQ ID NO:880, SEQ IDNO:882, SEQ ID NO:884, SEQ ID NO:886, SEQ ID NO:888, SEQ ID NO:890, SEQID NO:892, SEQ ID NO:894, SEQ ID NO:896, SEQ ID NO:898, SEQ ID NO:900,SEQ ID NO:902, SEQ ID NO:904, SEQ ID NO:906, SEQ ID NO:908, SEQ IDNO:910, SEQ ID NO:912, SEQ ID NO:914, SEQ ID NO:916, SEQ ID NO:918, SEQID NO:920, SEQ ID NO:922, SEQ ID NO:924, SEQ ID NO:926, SEQ ID NO:928,SEQ ID NO:930, SEQ ID NO:932, SEQ ID NO:934, SEQ ID NO:936, SEQ IDNO:938, SEQ ID NO:940, SEQ ID NO:942, SEQ ID NO:944, SEQ ID NO:946, SEQID NO:948, SEQ ID NO:950, SEQ ID NO:952 or SEQ ID NO:954. As discussedabove, the identity can be over the full length of the polypeptide, or,the identity can be over a region of at least about 50, 60, 70, 80, 90,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or moreresidues. Polypeptides of the invention can also be shorter than thefull length of exemplary polypeptides. In one aspect, the inventionprovides a polypeptide comprising only a subsequence of a sequence ofthe invention, exemplary subsequences can be about 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues.In alternative aspects, the invention provides polypeptides (peptides,fragments) ranging in size between about 5 and the full length of apolypeptide, e.g., an enzyme, such as a hydrolase, including anesterase, an acylase, a lipase, a phospholipase or a protease; exemplarysizes being of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, or more residues, e.g., contiguous residues ofan exemplary hydrolase of the invention. Peptides of the invention canbe useful as, e.g., labeling probes, antigens, toleragens, motifs,hydrolase active sites. Polypeptides of the invention also includeantibodies capable of binding to a hydrolase of the invention.

Additionally, the invention provides isolated, synthetic or recombinantpolypeptides having a sequence identity (e.g., at least 50% sequenceidentity) to an exemplary sequence of the invention, e.g., SEQ IDNO:956. SEQ ID NO:958, SEQ ID NO:960, SEQ ID NO:962, SEQ ID NO:964, SEQID NO:966. SEQ ID NO:968, SEQ ID NO:970, SEQ ID NO:972, SEQ ID NO:974,SEQ ID NO:976, SEQ ID NO:978, SEQ ID NO:980, SEQ ID NO:982, or SEQ IDNO:984.

The polypeptides of the invention include hydrolases in an active orinactive form. For example, the polypeptides of the invention includeproproteins before “maturation” or processing of prepro sequences, e.g.,by a proprotein-processing enzyme, such as a proprotein convertase togenerate an “active” mature protein. The polypeptides of the inventioninclude hydrolases inactive for other reasons, e.g., before “activation”by a post-translational processing event, e.g., an endo- orexo-peptidase or proteinase action, a phosphorylation event, anamidation, a glycosylation or a sulfation, a dimerization event, and thelike. Methods for identifying “prepro” domain sequences and signalsequences are well known in the art, see, e.g., Van de Ven (1993) Crit.Rev. Oncog. 4(2):115-136. For example, to identify a prepro sequence,the protein is purified from the extracellular space and the N-terminalprotein sequence is determined and compared to the unprocessed form.

The polypeptides of the invention include all active forms, includingactive subsequences, e.g., catalytic domains or active sites, of anenzyme of the invention. In one aspect, the invention provides catalyticdomains or active sites as set forth below. In one aspect, the inventionprovides a peptide or polypeptide comprising or consisting of an activesite domain as predicted through use of a database such as Pfam (whichis a large collection of multiple sequence alignments and hidden Markovmodels covering many common protein families, The Pfam protein familiesdatabase, A. Bateman, E. Birney, L. Cerruti, R. Durbin, L. Etwiller, S.R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall, and E. L. L.Sonnhammer, Nucleic Acids Research, 30(1):276-280, 2002) or equivalent.

The invention includes polypeptides with or without a signal sequenceand/or a prepro sequence. The invention includes polypeptides withheterologous signal sequences and/or prepro sequences. The preprosequence (including a sequence of the invention used as a heterologousprepro domain) can be located on the amino terminal or the carboxyterminal end of the protein. The invention also includes isolated orrecombinant signal sequences, prepro sequences and catalytic domains(e.g., “active sites”) comprising sequences of the invention.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, in one aspect, amimetic composition is within the scope of the invention if it has ahydrolase activity.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2, 3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide Aspartyl or glutamylcan also be converted to asparaginyl and glutaminyl residues by reactionwith ammonium ions. Mimetics of basic amino acids can be generated bysubstitution with, e.g., (in addition to lysine and arginine) the aminoacids ornithine, citrulline, or (guanidino)-acetic acid, or(guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrilederivative (e.g., containing the CN-moiety in place of COOH) can besubstituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R- or S-form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T.E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptides, or fragments thereof, of the invention.Such method have been known in the art since the early 1960's(Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (See alsoStewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd Ed.,Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have recently beenemployed in commercially available laboratory peptide design andsynthesis kits (Cambridge Research Biochemicals). Such commerciallyavailable laboratory kits have generally utilized the teachings of H. M.Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and providefor synthesizing peptides upon the tips of a multitude of “rods” or“pins” all of which are connected to a single plate. When such a systemis utilized, a plate of rods or pins is inverted and inserted into asecond plate of corresponding wells or reservoirs, which containsolutions for attaching or anchoring an appropriate amino acid to thepin's or rod's tips. By repeating such a process step, i.e., invertingand inserting the rod's and pin's tips into appropriate solutions, aminoacids are built into desired peptides. In addition, a number ofavailable FMOC peptide synthesis systems are available. For example,assembly of a polypeptide or fragment can be carried out on a solidsupport using an Applied Biosystems, Inc. Model 431A™ automated peptidesynthesizer. Such equipment provides ready access to the peptides of theinvention, either by direct synthesis or by synthesis of a series offragments that can be coupled using other known techniques.

Enzymes of the Invention

The invention provides novel hydrolases, including esterases, acylases,lipases, phospholipases or proteases, e.g., proteins comprising at leastabout 50% sequence identity to an exemplary polypeptide of theinvention, e.g., a protein having a sequence as set forth in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, etc., antibodies that bindthem, and methods for making and using them. The polypeptides of theinvention can have any hydrolase activity, e.g., an esterase, acylase,lipase, phospholipase or protease activity. In alternative aspects, anactivity of an enzyme of the invention comprises hydrolysis or synthesisof lipids or oils. The hydrolases of the invention can modify oils byhydrolysis, alcoholysis, esterification, transesterification and/orinteresterification, including “forced migration” reactions.

In alternative aspects, the hydrolases of the invention can havemodified or new activities as compared to the exemplary hydrolases orthe activities described herein. For example, the invention includeshydrolases with and without signal sequences and the signal sequencesthemselves. The invention includes immobilized hydrolases,anti-hydrolase antibodies and fragments thereof. The invention providesproteins for inhibiting hydrolase activity, e.g., antibodies that bindto the hydrolase active site. The invention includes homodimers andheterocomplexes, e.g., fusion proteins, heterodimers, etc., comprisingthe hydrolases of the invention. The invention includes hydrolaseshaving activity over a broad range of high and low temperatures and pH's(e.g., acidic and basic aqueous conditions).

In one aspect, one or more hydrolases (e.g., lipases) of the inventionis used for the biocatalytic synthesis of structured lipids, i.e.,lipids that contain a defined set of fatty acids distributed in adefined manner on the glycerol backbone, including cocoa butteralternatives, poly-unsaturated fatty acids (PUFAs), 1,3-diacylglycerides (DAGs), 2-monoglycerides (MAGs) and triacylglycerides (TAGs).

In one aspect, the invention provides methods of generating enzymeshaving altered (higher or lower) K_(cat)/K_(m). In one aspect,site-directed mutagenesis is used to create additional hydrolase enzymeswith alternative substrate specificities. The can be done, for example,by redesigning the substrate binding region or the active site of theenzyme. In one aspect, hydrolases of the invention are more stable athigh temperatures, such as 80° C. to 85° C. to 90° C. to 95° C., ascompared to hydrolases from conventional or moderate organisms.

Various proteins of the invention have a hydrolase activity, e.g., anesterase, acylase, lipase, phospholipase or protease activity, undervarious conditions. The invention provides methods of making hydrolaseswith different catalytic efficiency and stabilities towards temperature,oxidizing agents and pH conditions. These methods can use, e.g., thetechniques of site-directed mutagenesis and/or random mutagenesis. Inone aspect, directed evolution can be used to produce hydrolases withalternative specificities and stability.

The proteins of the invention are used in methods of the invention thatcan identify hydrolase modulators, e.g., activators or inhibitors.Briefly, test samples (e.g., compounds, such as members of peptide orcombinatorial libraries, broths, extracts, and the like) are added tohydrolase assays to determine their ability to modulate, e.g., inhibitor activate, substrate cleavage. These inhibitors can be used inindustry and research to reduce or prevent undesired isomerization.Modulators found using the methods of the invention can be used to alter(e.g., decrease or increase) the spectrum of activity of a hydrolase.

The invention also provides methods of discovering hydrolases using thenucleic acids, polypeptides and antibodies of the invention. In oneaspect, lambda phage libraries are screened for expression-baseddiscovery of hydrolases. In one aspect, the invention uses lambda phagelibraries in screening to allow detection of toxic clones; improvedaccess to substrate; reduced need for engineering a host, by-passing thepotential for any bias resulting from mass excision of the library; and,faster growth at low clone densities. Screening of lambda phagelibraries can be in liquid phase or in solid phase. In one aspect, theinvention provides screening in liquid phase. This gives a greaterflexibility in assay conditions; additional substrate flexibility;higher sensitivity for weak clones; and ease of automation over solidphase screening.

The invention provides screening methods using the proteins and nucleicacids of the invention involving robotic automation. This enables theexecution of many thousands of biocatalytic reactions and screeningassays in a short period of time, e.g., per day, as well as ensuring ahigh level of accuracy and reproducibility (see discussion of arrays,below). As a result, a library of derivative compounds can be producedin a matter of weeks.

The invention includes hydrolase enzymes which are non-naturallyoccurring hydrolases having a different hydrolase activity, stability,substrate specificity, pH profile and/or performance characteristic ascompared to the non-naturally occurring hydrolase. These hydrolases havean amino acid sequence not found in nature. They can be derived bysubstitution of a plurality of amino acid residues of a precursorhydrolase with different amino acids. The precursor hydrolase may be anaturally-occurring hydrolase or a recombinant hydrolase. In one aspect,the hydrolase variants encompass the substitution of any of thenaturally occurring L-amino acids at the designated amino acid residuepositions.

Hydrolase Signal Sequences, Prepro and Catalytic Domains

The invention provides signal sequences (e.g., signal peptides (SPs)),prepro domains and catalytic domains (CDs). The SPs, prepro domainsand/or CDs of the invention can be isolated or recombinant peptides orcan be part of a fusion protein, e.g., as a heterologous domain in achimeric protein. The invention provides nucleic acids encoding thesecatalytic domains (CDs), prepro domains and signal sequences (SPs, e.g.,a peptide having a sequence comprising/consisting of amino terminalresidues of a polypeptide of the invention). In one aspect, theinvention provides a signal sequence comprising a peptidecomprising/consisting of a sequence as set forth in residues 1 to 12, 1to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44 (ora longer peptide) of a polypeptide of the invention.

The hydrolase signal sequences (SPs), CDs, and/or prepro sequences ofthe invention can be isolated peptides, or, sequences joined to anotherhydrolase or a non-hydrolase polypeptide, e.g., as a fusion (chimeric)protein. In one aspect, the invention provides polypeptides comprisinghydrolase signal sequences of the invention. In one aspect, polypeptidescomprising hydrolase signal sequences SPs, CDs, and/or prepro of theinvention comprise sequences heterologous to hydrolases of the invention(e.g., a fusion protein comprising an SP, CD, and/or prepro of theinvention and sequences from another hydrolase or a non-hydrolaseprotein). In one aspect, the invention provides hydrolases of theinvention with heterologous SPs, CDs, and/or prepro sequences, e.g.,sequences with a yeast signal sequence. A hydrolase of the invention cancomprise a heterologous SP and/or prepro in a vector, e.g., a pPICseries vector (Invitrogen, Carlsbad, Calif.).

In one aspect, SPs, CDs, and/or prepro sequences of the invention areidentified following identification of novel hydrolase polypeptides. Thepathways by which proteins are sorted and transported to their propercellular location are often referred to as protein targeting pathways.One of the most important elements in all of these targeting systems isa short amino acid sequence at the amino terminus of a newly synthesizedpolypeptide called the signal sequence. This signal sequence directs aprotein to its appropriate location in the cell and is removed duringtransport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. The signal sequences can vary in length from 13to 45 or more amino acid residues. Various methods of recognition ofsignal sequences are known to those of skill in the art. For example, inone aspect, novel hydrolase signal peptides are identified by a methodreferred to as SignalP. SignalP uses a combined neural network whichrecognizes both signal peptides and their cleavage sites. (Nielsen, etal., “Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites.” Protein Engineering, vol. 10, no.1, p. 1-6 (1997).

It should be understood that in some aspects hydrolases of the inventionmay not have SPs and/or prepro sequences, and/or catalytic domains(CDs). In one aspect, the invention provides polypeptides (e.g.,hydrolases) lacking all or part of an SP, a CD and/or a prepro domain.In one aspect, the invention provides a nucleic acid sequence encoding asignal sequence (SP), a CD, and/or prepro from one hydrolase operablylinked to a nucleic acid sequence of a different hydrolase or,optionally, a signal sequence (SPs) and/or prepro domain from anon-hydrolase protein may be desired.

The invention also provides isolated or recombinant polypeptidescomprising signal sequences (SPs), prepro domain and/or catalyticdomains (CDs) of the invention and heterologous sequences. Theheterologous sequences are sequences not naturally associated (e.g., toa hydrolase) with an SP, prepro domain and/or CD. The sequence to whichthe SP, prepro domain and/or CD are not naturally associated can be onthe SP's, prepro domain and/or CD's amino terminal end, carboxy terminalend, and/or on both ends of the SP and/or CD. In one aspect, theinvention provides an isolated or recombinant polypeptide comprising (orconsisting of) a polypeptide comprising a signal sequence (SP), preprodomain and/or catalytic domain (CD) of the invention with the provisothat it is not associated with any sequence to which it is naturallyassociated (e.g., hydrolase sequence). Similarly in one aspect, theinvention provides isolated or recombinant nucleic acids encoding thesepolypeptides. Thus, in one aspect, the isolated or recombinant nucleicacid of the invention comprises coding sequence for a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention and aheterologous sequence (i.e., a sequence not naturally associated withthe a signal sequence (SP), prepro domain and/or catalytic domain (CD)of the invention). The heterologous sequence can be on the 3′ terminalend, 5′ terminal end, and/or on both ends of the SP, prepro domainand/or CD coding sequence.

The invention provides fusion of N-terminal or C-terminal subsequencesof enzymes of the invention (e.g., signal sequences, prepro sequences)with other polypeptides, active proteins or protein fragments. Theproduction of an enzyme of the invention (e.g., a hydrolase, e.g., alipase such as a phospholipase) may also be accomplished by expressingthe enzyme as an inactive fusion protein that is later activated by aproteolytic cleavage event (using either an endogenous or exogenousprotease activity, e.g. trypsin) that results in the separation of thefusion protein partner and the mature enzyme, e.g., hydrolase of theinvention. In one aspect, the fusion protein of the invention isexpressed from a hybrid nucleotide construct that encodes a single openreading frame containing the following elements: the nucleotide sequencefor the fusion protein, a linker sequence (defined as a nucleotidesequence that encodes a flexible amino acid sequence that joins two lessflexible protein domains), protease cleavage recognition site, and themature enzyme (e.g., any enzyme of the invention, e.g., a hydrolase)sequence. In alternative aspects, the fusion protein can comprise apectate lyase sequence, a xylanase sequence, a phosphatidic acidphosphatase sequence, or another sequence, e.g., a sequence that haspreviously been shown to be over-expressed in a host system of interest.Any host system can be used (see discussion, above), for example, E.coli or Pichia pastoris. The arrangement of the nucleotide sequences inthe chimeric nucleotide construction can be determined based on theprotein expression levels achieved with each fusion construct.Proceeding from the 5′ end of the nucleotide construct to the 3′ primeend of the construct, in one aspect, the nucleotide sequences isassembled as follows: Signal sequence/fusion protein/linkersequence/protease cleavage recognition site/mature enzyme (e.g., anyenzyme of the invention, e.g., a hydrolase) or Signal sequence/prosequence/mature enzyme/linker sequence/fusion protein. The expression ofenzyme (e.g., any enzyme of the invention, e.g., a hydrolase) as aninactive fusion protein may improve the overall expression of theenzyme's sequence, may reduce any potential toxicity associated with theoverproduction of active enzyme and/or may increase the shelf life ofenzyme prior to use because enzyme would be inactive until the fusionprotein e.g. pectate lyase is separated from the enzyme, e.g., hydrolaseof the invention.

In various aspects, the invention provides specific formulations for theactivation of a hydrolase of the invention expressed as a fusionprotein. In one aspect, the activation of the hydrolase activityinitially expressed as an inactive fusion protein is accomplished usinga proteolytic activity or potentially a proteolytic activity incombination with an amino-terminal or carboxyl-terminal peptidase (thepeptidase can be an enzyme of the invention, or, another enzyme). Thisactivation event may be accomplished in a variety of ways and at varietyof points in the manufacturing/storage process prior to application inoil degumming Exemplary processes of the invention include: Cleavage byan endogenous activity expressed by the manufacturing host uponsecretion of the fusion construct into the fermentation media; Cleavageby an endogenous protease activity (which can be a protease of theinvention) that is activated or comes in contact with intracellularlyexpressed fusion construct upon rupture of the host cells; Passage ofthe crude or purified fusion construct over a column of immobilizedprotease (which can be a protease of the invention) activity toaccomplish cleavage and enzyme (e.g., hydrolase of the invention, e.g.,a protease, lipase, esterase or phospholipase) activation prior toenzyme formulation; Treatment of the crude or purified fusion constructwith a soluble source of proteolytic activity; Activation of a hydrolase(e.g., a hydrolase of the invention) at the oil refinery using either asoluble or insoluble source of proteolytic activity immediately prior touse in the process; and/or, Activation of the hydrolase (e.g., ahydrolase of the invention) activity by continuously circulating thefusion construct formulation through a column of immobilized proteaseactivity at reduced temperature (for example, any between about 4° C.and 20° C.). This activation event may be accomplished prior to deliveryto the site of use or it may occur on-site at the oil refinery.

Glycosylation

The peptides and polypeptides of the invention (e.g., hydrolases,antibodies) can also be glycosylated, for example, in one aspect,comprising at least one glycosylation site, e.g., an N-linked orO-linked glycosylation. In one aspect, the polypeptide can beglycosylated after being expressed in a P. pastoris or a S. pombe. Theglycosylation can be added post-translationally either chemically or bycellular biosynthetic mechanisms, wherein the later incorporates the useof known glycosylation motifs, which can be native to the sequence orcan be added as a peptide or added in the nucleic acid coding sequence.

Assays for Phospholipase Activity

The invention provides isolated or recombinant polypeptides having aphospholipase activity and nucleic acids encoding them. Any of the manyphospholipase activity assays known in the art can be used to determineif a polypeptide has a phospholipase activity and is within the scope ofthe invention. Routine protocols for determining phospholipase A, B, Dand C, patatin and lipid acyl hydrolase activities are well known in theart.

Exemplary activity assays include turbidity assays, methylumbelliferylphosphocholine (fluorescent) assays, Amplex red (fluorescent)phospholipase assays, thin layer chromatography assays (TLC), cytolyticassays and p-nitrophenylphosphorylcholine assays. Using these assayspolypeptides can be quickly screened for phospholipase activity.

The phospholipase activity can comprise a lipid acyl hydrolase (LAH)activity. See, e.g., Jimenez (2001) Lipids 36:1169-1174, describing anoctaethylene glycol monododecyl ether-based mixed micellar assay fordetermining the lipid acyl hydrolase activity of a patatin. Pinsirodom(2000) J. Agric. Food Chem. 48:155-160, describes an exemplary lipidacyl hydrolase (LAH) patatin activity.

Turbidity assays to determine phospholipase activity are described,e.g., in Kauffmann (2001) “Conversion of Bacillus thermocatenulatuslipase into an efficient phospholipase with increased activity towardslong-chain fatty acyl substrates by directed evolution and rationaldesign,” Protein Engineering 14:919-928; Ibrahim (1995) “Evidenceimplicating phospholipase as a virulence factor of Candida albicans,”Infect. Immun 63:1993-1998.

Methylumbelliferyl (fluorescent) phosphocholine assays to determinephospholipase activity are described, e.g., in Goode (1997) “Evidencefor cell surface and internal phospholipase activity in ascidian eggs,”Develop. Growth Differ. 39:655-660; Diaz (1999) “Directfluorescence-based lipase activity assay,” BioTechniques 27:696-700.

Amplex Red (fluorescent) Phospholipase Assays to determine phospholipaseactivity are available as kits, e.g., the detection ofphosphatidylcholine-specific phospholipase using an Amplex Redphosphatidylcholine-specific phospholipase assay kit from MolecularProbes Inc. (Eugene, Oreg.), according to manufacturer's instructions.Fluorescence is measured in a fluorescence microplate reader usingexcitation at 560±10 nm and fluorescence detection at 590±10 nm. Theassay is sensitive at very low enzyme concentrations.

Thin layer chromatography assays (TLC) to determine phospholipaseactivity are described, e.g., in Reynolds (1991) Methods in Enzymol.197:3-13; Taguchi (1975) “Phospholipase from Clostridium novyi typeA.I,” Biochim Biophys. Acta 409:75-85. Thin layer chromatography (TLC)is a widely used technique for detection of phospholipase activity.Various modifications of this method have been used to extract thephospholipids from the aqueous assay mixtures. In some PLC assays thehydrolysis is stopped by addition of chloroform/methanol (2:1) to thereaction mixture. The unreacted starting material and the diacylglycerolare extracted into the organic phase and may be fractionated by TLC,while the head group product remains in the aqueous phase. For moreprecise measurement of the phospholipid digestion, radiolabeledsubstrates can be used (see, e.g., Reynolds (1991) Methods in Enzymol.197:3-13). The ratios of products and reactants can be used to calculatethe actual number of moles of substrate hydrolyzed per unit time. If allthe components are extracted equally, any losses in the extraction willaffect all components equally. Separation of phospholipid digestionproducts can be achieved by silica gel TLC withchloroform/methanol/water (65:25:4) used as a solvent system (see, e.g.,Taguchi (1975) Biochim Biophys. Acta 409:75-85).

p-Nitrophenylphosphorylcholine assays to determine phospholipaseactivity are described, e.g., in Korbsrisate (1999) J. Clin. Microbiol.37:3742-3745; Berka (1981) Infect. Immun. 34:1071-1074. This assay isbased on enzymatic hydrolysis of the substrate analogp-nitrophenylphosphorylcholine to liberate a yellow chromogenic compoundp-nitrophenol, detectable at 405 nm. This substrate is convenient forhigh-throughput screening.

A cytolytic assay can detect phospholipases with cytolytic activitybased on lysis of erythrocytes. Toxic phospholipases can interact witheukaryotic cell membranes and hydrolyze phosphatidylcholine andsphingomyelin, leading to cell lysis. See, e.g., Titball (1993)Microbiol. Rev. 57:347-366.

Hybrid Hydrolases and Peptide Libraries

In one aspect, the invention provides hybrid hydrolases and fusionproteins, including peptide libraries, comprising sequences of theinvention. The peptide libraries of the invention can be used to isolatepeptide modulators (e.g., activators or inhibitors) of targets. Thepeptide libraries of the invention can be used to identify formalbinding partners of targets, such as ligands, e.g., cytokines, hormonesand the like.

In one aspect, the fusion proteins of the invention (e.g., the peptidemoiety) are conformationally stabilized (relative to linear peptides) toallow a higher binding affinity for targets. The invention providesfusions of hydrolases of the invention and other peptides, includingknown and random peptides. They can be fused in such a manner that thestructure of the hydrolases are not significantly perturbed and thepeptide is metabolically or structurally conformationally stabilized.This allows the creation of a peptide library that is easily monitoredboth for its presence within cells and its quantity.

Amino acid sequence variants of the invention can be characterized by apredetermined nature of the variation, a feature that sets them apartfrom a naturally occurring form, e.g, an allelic or interspeciesvariation of a hydrolase sequence. In one aspect, the variants of theinvention exhibit the same qualitative biological activity as thenaturally occurring analogue. Alternatively, the variants can beselected for having modified characteristics. In one aspect, while thesite or region for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed hydrolase variants screened for the optimalcombination of desired activity. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, as discussed herein for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants can be done using assays ofproteolytic activities. In alternative aspects, amino acid substitutionscan be single residues; insertions can be on the order of from about 1to 20 amino acids, although considerably larger insertions can be done.Deletions can range from about 1 to about 20, 30, 40, 50, 60, 70residues or more. To obtain a final derivative with the optimalproperties, substitutions, deletions, insertions or any combinationthereof may be used. Generally, these changes are done on a few aminoacids to minimize the alteration of the molecule. However, largerchanges may be tolerated in certain circumstances.

The invention provides hydrolases where the structure of the polypeptidebackbone, the secondary or the tertiary structure, e.g., analpha-helical or beta-sheet structure, has been modified. In one aspect,the charge or hydrophobicity has been modified. In one aspect, the bulkof a side chain has been modified. Substantial changes in function orimmunological identity are made by selecting substitutions that are lessconservative. For example, substitutions can be made which moresignificantly affect: the structure of the polypeptide backbone in thearea of the alteration, for example an alpha-helical or a beta-sheetstructure; a charge or a hydrophobic site of the molecule, which can beat an active site; or a side chain. The invention provides substitutionsin polypeptide of the invention where (a) a hydrophilic residues, e.g.seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g. glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine. The variants can exhibit the same qualitative biologicalactivity (i.e. hydrolase activity) although variants can be selected tomodify the characteristics of the hydrolases as needed.

In one aspect, hydrolases of the invention comprise epitopes orpurification tags, signal sequences or other fusion sequences, etc. Inone aspect, the hydrolases of the invention can be fused to a randompeptide to form a fusion polypeptide. By “fused” or “operably linked”herein is meant that the random peptide and the hydrolase are linkedtogether, in such a manner as to minimize the disruption to thestability of the hydrolase structure, e.g., it retains hydrolaseactivity. The fusion polypeptide (or fusion polynucleotide encoding thefusion polypeptide) can comprise further components as well, includingmultiple peptides at multiple loops.

In one aspect, the peptides (e.g., hydrolase subsequences) and nucleicacids encoding them are randomized, either fully randomized or they arebiased in their randomization, e.g. in nucleotide/residue frequencygenerally or per position. “Randomized” means that each nucleic acid andpeptide consists of essentially random nucleotides and amino acids,respectively. In one aspect, the nucleic acids which give rise to thepeptides can be chemically synthesized, and thus may incorporate anynucleotide at any position. Thus, when the nucleic acids are expressedto form peptides, any amino acid residue may be incorporated at anyposition. The synthetic process can be designed to generate randomizednucleic acids, to allow the formation of all or most of the possiblecombinations over the length of the nucleic acid, thus forming a libraryof randomized nucleic acids. The library can provide a sufficientlystructurally diverse population of randomized expression products toaffect a probabilistically sufficient range of cellular responses toprovide one or more cells exhibiting a desired response. Thus, theinvention provides an interaction library large enough so that at leastone of its members will have a structure that gives it affinity for somemolecule, protein, or other factor.

Screening Methodologies and “On-line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides forhydrolase activity, to screen compounds as potential activators orinhibitors of a hydrolase activity (e.g., for potential drug screening),for antibodies that bind to a polypeptide of the invention, for nucleicacids that hybridize to a nucleic acid of the invention, to screen forcells expressing a polypeptide of the invention and the like. See, e.g.,U.S. Pat. No. 6,337,187.

Capillary Arrays

Capillary arrays, such as the GIGAMATRIX™, Diversa Corporation, SanDiego, Calif., can be used to in the methods of the invention. Nucleicacids or polypeptides of the invention can be immobilized to or appliedto an array, including capillary arrays. Arrays can be used to screenfor or monitor libraries of compositions (e.g., small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide of theinvention. Capillary arrays provide another system for holding andscreening samples. For example, a sample screening apparatus can includea plurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The apparatus can further include interstitialmaterial disposed between adjacent capillaries in the array, and one ormore reference indicia formed within of the interstitial material. Acapillary for screening a sample, wherein the capillary is adapted forbeing bound in an array of capillaries, can include a first walldefining a lumen for retaining the sample, and a second wall formed of afiltering material, for filtering excitation energy provided to thelumen to excite the sample.

A polypeptide or nucleic acid, e.g., a ligand or a substrate, can beintroduced into a first component into at least a portion of a capillaryof a capillary array. Each capillary of the capillary array can compriseat least one wall defining a lumen for retaining the first component. Anair bubble can be introduced into the capillary behind the firstcomponent. A second component can be introduced into the capillary,wherein the second component is separated from the first component bythe air bubble. A sample of interest can be introduced as a first liquidlabeled with a detectable particle into a capillary of a capillaryarray, wherein each capillary of the capillary array comprises at leastone wall defining a lumen for retaining the first liquid and thedetectable particle, and wherein the at least one wall is coated with abinding material for binding the detectable particle to the at least onewall. The method can further include removing the first liquid from thecapillary tube, wherein the bound detectable particle is maintainedwithin the capillary, and introducing a second liquid into the capillarytube.

The capillary array can include a plurality of individual capillariescomprising at least one outer wall defining a lumen. The outer wall ofthe capillary can be one or more walls fused together. Similarly, thewall can define a lumen that is cylindrical, square, hexagonal or anyother geometric shape so long as the walls form a lumen for retention ofa liquid or sample. The capillaries of the capillary array can be heldtogether in close proximity to form a planar structure. The capillariescan be bound together, by being fused (e.g., where the capillaries aremade of glass), glued, bonded, or clamped side-by-side. The capillaryarray can be formed of any number of individual capillaries, forexample, a range from 100 to 4,000,000 capillaries. A capillary arraycan form a micro titer plate having about 100,000 or more individualcapillaries bound together.

Arrays, or “Biochips”

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. For example, in oneaspect of the invention, a monitored parameter is transcript expressionof a hydrolase gene. One or more, or, all the transcripts of a cell canbe measured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the invention. Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins. The present invention can be practiced with any known “array,”also referred to as a “microarray” or “nucleic acid array” or“polypeptide array” or “antibody array” or “biochip,” or variationthereof. Arrays are generically a plurality of “spots” or “targetelements,” each target element comprising a defined amount of one ormore biological molecules, e.g., oligonucleotides, immobilized onto adefined area of a substrate surface for specific binding to a samplemolecule, e.g., mRNA transcripts.

In one aspect, the hydrolases are used as immobilized forms. Anyimmobilization method can be used, e.g., immobilization upon an inertsupport such as diethylaminoethyl-cellulose, porous glass, chitin orcells. Cells that express hydrolases of the invention can be immobilizedby cross-linking, e.g. with glutaraldehyde to a substrate surface.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated or recombinant antibodies thatspecifically bind to a hydrolase of the invention. These antibodies canbe used to isolate, identify or quantify the hydrolase of the inventionor related polypeptides. These antibodies can be used to isolate otherpolypeptides within the scope the invention or other related hydrolases.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the invention.Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, NY (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

Polypeptides or peptides can be used to generate antibodies, which bindspecifically to the polypeptides of the invention. The resultingantibodies may be used in immunoaffinity chromatography procedures toisolate or purify the polypeptide or to determine whether thepolypeptide is present in a biological sample. In such procedures, aprotein preparation, such as an extract, or a biological sample iscontacted with an antibody capable of specifically binding to one of thepolypeptides of the invention.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of the invention.After a wash to remove non-specifically bound proteins, the specificallybound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of theinvention can be obtained by direct injection of the polypeptides intoan animal or by administering the polypeptides to a non-human animal.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique, the trioma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (see,e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to the polypeptides of the invention. Alternatively,transgenic mice may be used to express humanized antibodies to thesepolypeptides or fragments thereof.

Antibodies generated against the polypeptides of the invention(including anti-idiotype antibodies) may be used in screening forsimilar polypeptides from other organisms and samples. In suchtechniques, polypeptides from the organism are contacted with theantibody and those polypeptides which specifically bind the antibody aredetected. Any of the procedures described above may be used to detectantibody binding.

Immobilized Hydrolases

In one aspect, the hydrolase of the invention, e.g., esterases,acylases, lipases, phospholipases or proteases, are used as immobilizedforms, e.g., to process lipids, in the structured synthesis of lipids,to digest proteins and the like. The immobilized lipases of theinvention can be used, e.g., for hydrolysis of triglycerides,diglycerides or esters or for the esterification or transesterificationof fatty acids, diglycerides or triglycerides, or in theinteresterification of fats. In one aspect, the lipase is specific foresterification of fatty acids with alcohol, 1,3-specific or randomizingtransesterification lipase or lipase specific for the hydrolysis ofpartial glycerides, esters or triglycerides Immobilized lipase of theinvention can be used in a packed bed for continuous transesterificationof solvent free fats. See, e.g., U.S. Pat. Nos. 4,818,695; 5,569,594.

Any immobilization method or form of support can be used, e.g., arrays,beads, capillary supports and the like, as described above. In oneaspect, hydrolase immobilization can occur upon an inert support such asdiethylaminoethyl-cellulose, porous glass, chitin or cells. Cells thatexpress hydrolases of the invention can be immobilized by cross-linking,e.g. with glutaraldehyde to a substrate surface. Immobilized hydrolasesof the invention can be prepared containing hydrolase bound to a dry,porous particulate hydrophobic support, with a surfactant, such as apolyoxyethylene sorbitan fatty acid ester or a polyglycerol fatty acidester. The support can be an aliphatic olefinic polymer, such as apolyethylene or a polypropylene, a homo- or copolymer of styrene or ablend thereof or a pre-treated inorganic support. These supports can beselected from aliphatic olefinic polymers, oxidation polymers, blends ofthese polymers or pre-treated inorganic supports in order to make thesesupports hydrophobic. This pre-treatment can comprise silanization withan organic silicon compound. The inorganic material can be a silica, analumina, a glass or a ceramic. Supports can be made from polystyrene,copolymers of styrene, polyethylene, polypropylene or from co-polymersderived from (meth)acrylates. See, e.g., U.S. Pat. No. 5,773,266.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, transgenic seeds or plantsor plant parts, polypeptides (e.g., hydrolases) and/or antibodies of theinvention. The kits also can contain instructional material teaching themethodologies and industrial uses of the invention, as described herein.

Measuring Metabolic Parameters

The methods of the invention provide whole cell evolution, or whole cellengineering, of a cell to develop a new cell strain having a newphenotype by modifying the genetic composition of the cell, where thegenetic composition is modified by addition to the cell of a nucleicacid, e.g., a hydrolase-encoding nucleic acid of the invention. Todetect the new phenotype, at least one metabolic parameter of a modifiedcell is monitored in the cell in a “real time” or “on-line” time frame.In one aspect, a plurality of cells, such as a cell culture, ismonitored in “real time” or “on-line.” In one aspect, a plurality ofmetabolic parameters is monitored in “real time” or “on-line.” Metabolicparameters can be monitored using the fluorescent polypeptides of theinvention (e.g., hydrolases of the invention comprising a fluorescentmoiety).

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods of the invention, metabolic networks are established, includingthe:

-   -   identity of all pathway substrates, products and intermediary        metabolites    -   identity of all the chemical reactions interconverting the        pathway metabolites, the stoichiometry of the pathway reactions,    -   identity of all the enzymes catalyzing the reactions, the enzyme        reaction kinetics,    -   the regulatory interactions between pathway components, e.g.        allosteric interactions, enzyme-enzyme interactions etc,    -   intracellular compartmentalization of enzymes or any other        supramolecular organization of the enzymes, and,    -   the presence of any concentration gradients of metabolites,        enzymes or effector molecules or diffusion barriers to their        movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In oneaspect of the methods of the invention, after the on-line MFAcalculation, the dynamic behavior of the cells, their phenotype andother properties are analyzed by investigating the pathway utilization.For example, if the glucose supply is increased and the oxygen decreasedduring the yeast fermentation, the utilization of respiratory pathwayswill be reduced and/or stopped, and the utilization of the fermentativepathways will dominate. Control of physiological state of cell cultureswill become possible after the pathway analysis. The methods of theinvention can help determine how to manipulate the fermentation bydetermining how to change the substrate supply, temperature, use ofinducers, etc. to control the physiological state of cells to move alongdesirable direction. In practicing the methods of the invention, the MFAresults can also be compared with transcriptome and proteome data todesign experiments and protocols for metabolic engineering or geneshuffling, etc.

In practicing the methods of the invention, any modified or newphenotype can be conferred and detected, including new or improvedcharacteristics in the cell. Any aspect of metabolism or growth can bemonitored.

Monitoring Expression of an mRNA Transcript

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript orgenerating new transcripts in a cell. This increased or decreasedexpression can be traced by use of a hydrolase-encoding nucleic acid ofthe invention. mRNA transcripts, or messages, also can be detected andquantified by any method known in the art, including, e.g., Northernblots, quantitative amplification reactions, hybridization to arrays,and the like. Quantitative amplification reactions include, e.g.,quantitative PCR, including, e.g., quantitative reverse transcriptionpolymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or“real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001) Br. J. Haematol.114:313-318; Xia (2001) Transplantation 72:907-914).

In one aspect of the invention, the engineered phenotype is generated byknocking out expression of a homologous gene. The gene's coding sequenceor one or more transcriptional control elements can be knocked out,e.g., promoters or enhancers. Thus, the expression of a transcript canbe completely ablated or only decreased.

In one aspect of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide or generatingnew polypeptides in a cell. This increased or decreased expression canbe traced by use of a hydrolase or an antibody of the invention.Polypeptides, peptides and amino acids also can be detected andquantified by any method known in the art, including, e.g., nuclearmagnetic resonance (NMR), spectrophotometry, radiography (proteinradiolabeling), electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, various immunological methods, e gimmunoprecipitation, immunodiffusion, immuno-electrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immuno-fluorescent assays, gel electrophoresis (e.g., SDS-PAGE),staining with antibodies, fluorescent activated cell sorter (FACS),pyrolysis mass spectrometry, Fourier-Transform Infrared Spectrometry,Raman spectrometry, GC-MS, and LC-Electrospray andcap-LC-tandem-electrospray mass spectrometries, and the like. Novelbioactivities can also be screened using methods, or variations thereof,described in U.S. Pat. No. 6,057,103. Furthermore, as discussed below indetail, one or more, or, all the polypeptides of a cell can be measuredusing a protein array.

Industrial and Medical Applications

The invention provides many industrial uses and medical applications forthe hydrolases (e.g., lipases, phospholipases, esterases, proteases) ofthe invention, and a few exemplary uses and compositions of theinvention are described below. The processes of the invention compriseconverting a non-hydratable phospholipid to a hydratable form, oildegumming, food processing, processing of oils from plants, fish, algaeand the like, to name just a few applications.

Phospholipases

The invention provides many industrial uses and medical applications forthe hydrolases, e.g., lipases and phospholipases of the invention, e.g.,phospholipases A, B, C and D. Methods of using phospholipase enzymes inindustrial applications are well known in the art. For example, thephospholipases and methods of the invention can be used for theprocessing of fats and oils as described, e.g., in JP Patent ApplicationPublication H6-306386, describing converting phospholipids present inthe oils and fats into water-soluble substances containing phosphoricacid groups.

Phospholipases of the invention can be used to process plant oils andphospholipids such as those derived from or isolated from rice bran,soy, canola, palm, cottonseed, corn, palm kernel, coconut, peanut,sesame, sunflower. Phospholipases of the invention can be used toprocess essential oils, e.g., those from fruit seed oils, e.g.,grapeseed, apricot, borage, etc. Phospholipases of the invention can beused to process oils and phospholipids in different forms, includingcrude forms, degummed, gums, wash water, clay, silica, soapstock, andthe like. The phospholipids of the invention can be used to process highphosphorous oils, fish oils, animal oils, plant oils, algae oils and thelike. In any aspect of the invention, any time a phospholipase C can beused, an alternative comprises use of a phospholipase D of the inventionand a phosphatase (e.g., using a PLD/phosphatase combination to improveyield in a high phosphorus oil, such as a soy bean oil).

In one aspect, the invention provides compositions and methods (whichcan comprise use of phospholipases of the invention) for oil degummingcomprising using varying amounts of acid and base without makingsoapstock. Using this aspect of the invention for oil degumming, acid(including phosphoric and/or citric) can be used to hydratenon-hydratable phospholipids in high phosphorous oils (including, e.g.,rice bran, soybean, canola, and sunflower). Once the phospholipids arehydrated, the pH of the aqueous phase can be raised using causticaddition: the amount of caustic added can create a favorable pH forenzyme activity but will not result in the formation of a significantsoapstock fraction in the oil. Because a soapstock is not formed, thefree fatty acids in the oil can be removed downstream, following thedegumming step, during bleaching and deodorization.

Phospholipases of the invention can be used to process and make edibleoils, biodiesel oils, liposomes for pharmaceuticals and cosmetics,structured phospholipids and structured lipids. Phospholipases of theinvention can be used in oil extraction. Phospholipases of the inventioncan be used to process and make various soaps.

The phospholipases of the invention can also be used to study thephosphoinositide (PI) signaling system; in the diagnosis, prognosis anddevelopment of treatments for bipolar disorders (see, e.g., Pandey(2002) Neuropsychopharmacology 26:216-228); as antioxidants; as modifiedphospholipids; as foaming and gelation agents; to generate angiogeniclipids for vascularizing tissues; to identify phospholipase, e.g., PLA,PLB, PLC, PLD and/or patatin modulators (agonists or antagonists), e.g.,inhibitors for use as anti-neoplastics, anti-inflammatory and asanalgesic agents. They can be used to generate acidic phospholipids forcontrolling the bitter taste in food and pharmaceuticals. They can beused in fat purification. They can be used to identify peptidesinhibitors for the treatment of viral, inflammatory, allergic andcardiovascular diseases. They can be used to make vaccines. They can beused to make polyunsaturated fatty acid glycerides andphosphatidylglycerols.

The phospholipases of the invention, for example PLA and PLC enzymes,are used to generate immunotoxins and various therapeutics foranti-cancer treatments.

The phospholipases of the invention can be used in conjunction withother enzymes for decoloring (i.e. chlorophyll removal) and indetergents (see above), e.g., in conjunction with other enzymes (e.g.,lipases, proteases, esterases, phosphatases). For example, in anyinstance where a PLC is used, a PLD and a phosphatase may be used incombination, to produce the same result as a PLC alone.

Detoxification

The hydrolases (e.g., lipases, esterase, protease and/or phospholipasesof the invention) can be used in detoxification processes, e.g., for thedetoxification of endotoxins, e.g., compositions comprisinglipopolysaccharides (LPS), and, the invention provides detoxificationprocesses using at least one enzyme of the invention, e.g., a hydrolasehaving a sequence as set forth in SEQ ID NO:962 (encoded by SEQ IDNO:961), or SEQ ID NO:966 (encoded by SEQ ID NO:965). In one aspect, alipase and/or an esterase of the invention is used to detoxify alipopolysaccharide (LPS). In one aspect, this detoxification is bydeacylation of 2′ and/or 3′ fatty acid chains from lipid A. In oneaspect, a hydrolase (e.g., a lipase and/or an esterase) of the inventionis used to hydrolyze a 2′-lauroyl and/or a 3′-myristoyl chain from alipid, e.g., a lipid A (e.g., from a bacterial endotoxin). In oneaspect, the process of the invention is used to destroy an endotoxin,e.g., a toxin from a gram negative bacteria, as from E. coli. In oneaspect, a hydrolase (e.g., a lipase and/or an esterase) of the inventionis used to ameliorate the effects of toxin poisoning (e.g., from anon-going gram negative infection), or, to prophylactically to preventthe effects of endotoxin during an infection (e.g., an infection in ananimal or a human). Accordingly, the invention provides a pharmaceuticalcomposition comprising a hydrolase (e.g., a lipase and/or an esterase)of the invention, and method using a hydrolase of the invention, for theamelioration or prevention of lipopolysaccharide (LPS) toxic effects,e.g., during sepsis.

Processing Foods

The hydrolases, e.g., lipases, esterases, proteases and/orphospholipases of the invention, or a combination thereof, can be usedto process foods, e.g., to change their stability, shelf-life, flavor,texture and the like. For example, in one aspect, phospholipases of theinvention are used to generate acidic phospholipids for controllingbitter taste in foods.

In one aspect, the invention provides cheese-making processes usinghydrolases (e.g., lipases, esterases, proteases, phospholipases) of theinvention (and, thus, the invention also provides cheeses comprisinghydrolases of the invention). In one aspect, the enzymes of theinvention (e.g., lipases, esterases, proteases, phospholipases, e.g.,phospholipase A, lysophospholipase or a combination thereof) are used toprocess cheeses for flavor enhancement, to increase yield and/or for“stabilizing” cheeses, e.g., by reducing the tendency for “oil-off,” or,in one aspect, the enzymes of the invention are used to produce cheesefrom cheese milk. These processes of the invention can incorporate anymethod or protocol, e.g., as described, e.g., in U.S. Pat. Nos.6,551,635, and 6,399,121, WO 03/070013, WO 00/054601. For example, inone aspect, hydrolases (e.g., lipases, esterases, proteases and/orphospholipases) of the invention are used to stabilize fat emulsion inmilk or milk-comprising compositions, e.g. cream, and are used tostabilize milk compositions, e.g. for the manufacturing of creams orcream liquors. In one aspect, the invention provides a process forenhancing the favor of a cheese using at least one enzyme of theinvention, the process comprising incubating a protein, a fat and aprotease (e.g., of the invention) and a lipase (e.g., of the invention)in an aqueous medium under conditions that produce an enhanced cheeseflavor (e.g., reduced bitterness), e.g., as described in WO 99/66805. Inone aspect, phospholipases of the invention are used to enhance flavorin a cheese (e.g., a curd) by mixing with water, a protease (e.g., ofthe invention), and a lipase (e.g., of the invention) at an elevatedtemperature, e.g., between about 75° C. to 95° C., as described, e.g.,in U.S. Pat. No. 4,752,483. In one aspect, phospholipases of theinvention are used to accelerate cheese aging by adding an enzyme of theinvention to a cheese (e.g., a cheese milk) before adding a coagulant tothe milk, or, adding an enzyme (e.g., a lipase or a phospholipase) ofthe invention to a curd with salt before pressing, e.g., as described,e.g., in U.S. Pat. No. 4,707,364. In one aspect, a lipase of theinvention is used degrade a triglyceride in milk fat to liberate freefatty acids, resulting in flavor enhancement. A protease of theinvention also can be used in any of these processes of the invention,see, e.g., Brindisi (2001) J. of Food Sci. 66:1100-1107.

In one aspect, a hydrolase (e.g., lipases, esterase, protease and/orphospholipase of the invention) is used to reduce the content ofphosphorus components in a food, e.g., an oil, such as a vegetable oilhaving a high non-hydratable phosphorus content, e.g., as described inWO 98/26057.

Caustic Refining

In one aspect, enzymes of the invention, e.g., phospholipases, lipases,esterases, proteases, are used as caustic refining aids. In one aspect,a PLC or PLD of the invention and a phosphatase are used in theprocesses as a drop-in, either before, during, or after a causticneutralization refining process (either continuous or batch refining.The amount of enzyme added may vary according to the process. The waterlevel used in the process should be low, e.g., about 0.5 to 5%.Alternatively, caustic is be added to the process multiple times. Inaddition, the process may be performed at different temperatures (25° C.to 70° C.), with different acids or caustics, and at varying pH (4-12).Acids that may be used in a caustic refining process include, but arenot limited to, phosphoric, citric, ascorbic, sulfuric, fumaric, maleic,hydrochloric and/or acetic acids. Acids are used to hydratenon-hydratable phospholipids. Caustics that may be used include, but arenot limited to, KOH— and NaOH. Caustics are used to neutralize freefatty acids. Alternatively, phospholipases of the invention, or moreparticularly a PLC or a PLD of the invention and a phosphatase, are usedfor purification of phytosterols from the gum/soapstock.

An alternate embodiment of the invention to add a phospholipase of theinvention before caustic refining, e.g., by expressing the phospholipasein a plant. In another embodiment, the phospholipase of the invention isadded during crushing of the plant, seeds or other plant part.Alternatively, the phospholipase of the invention is added followingcrushing, but prior to refining (i.e. in holding vessels). In addition,phospholipase is added as a refining pre-treatment, either with orwithout acid.

Another embodiment of the invention comprises adding a phospholipase ofthe invention during a caustic refining process. Levels of acid andcaustic can be varied depending on the level of phosphorous and thelevel of free fatty acids. Broad temperature and pH ranges can be usedin the process dependent upon the type of enzyme used.

In another embodiment of the invention, the phospholipase of theinvention is added after caustic refining. In one aspect, thephospholipase is added in an intense mixer or in a retention mixer,prior to separation. Alternatively, the phospholipase is added followingthe heat step. In another embodiment, the phospholipase of the inventionis added in the centrifugation step. In an additional embodiment, thephospholipase is added to the soapstock. Alternatively, thephospholipase is added to the washwater. In another instance, thephospholipase of the invention is added during the bleaching and/ordeodorizing steps.

Structured Synthesis and Processing of Oils

The invention provides methods for the structured synthesis of oils,lipids and the like using hydrolases (e.g., lipases, phospholipases,esterases, proteases) of the invention. The methods of the inventioncomprise a biocatalytic synthesis of structured lipids, i.e., lipidsthat contain a defined set of fatty acids distributed in a definedmanner on a backbone, e.g., a glycerol backbone. Products generatedusing the hydrolases of the invention and practicing the methods of theinvention include cocoa butter alternatives, lipids containingpoly-unsaturated fatty acids (PUFAs), 1,3-diacyl glycerides (DAGs),2-monoacylglycerides (MAGs) and triacylglycerides (TAGs). The methods ofthe invention enable synthesis of lipids or fatty acids with definedregiospecificities and stereoselectivities.

The invention provides methods for processing (modifying) oils, lipidsand the like using hydrolases of the invention. The methods of theinvention can be used to process oils from plants, animals,microorganisms. The methods of the invention can be used in thestructured synthesis of oils similar to those found in plants, animals,microorganisms. Lipids and oils can be processed to have a desiredcharacteristic. Lipids and oils that can be processed by the methods ofthe invention (using the hydrolases of the invention) include cocoabutter alternatives, lipids containing poly-unsaturated fatty acids(PUFAs), 1,3-diacyl glycerides (DAGs), 2-monoacylglycerides (MAGs) andtriacylglycerides (TAGs). In one aspect, the processed and syntheticoils and fats of the invention (e.g., cocoa butters alternatives andvegetable oils) can be used in a variety of applications, e.g., in theproduction of foods (e.g., confectionaries, pastries) and in theformulation of pharmaceuticals, nutraceuticals and cosmetics. In oneaspect, the invention provides methods of processing fats and oils,e.g., oilseeds, from plants, including, e.g., rice bran, canola,sunflower, olive, palm, soy or lauric type oils using a hydrolase, e.g.,a lipase, esterase or phospholipase, of the invention.

In one aspect, the invention provides methods of processing oils fromanimals, e.g., fish and mammals, using the hydrolases of the invention.In one aspect, the invention provides methods for the structuredsynthesis of oils similar to those found in animals, e.g., fish andmammals and microorganisms, using the hydrolases of the invention. Inone aspect, these synthetic or processes oils are used as feedadditives, foods, as ingredients in pharmaceutical formulations,nutraceuticals or in cosmetics. For example, in one aspect thehydrolases of the invention are used to make fish oil fatty acids as afeed additive. In one aspect, the hydrolases of the invention can beused to process oil from restaurant waste and rendered animal fats.

In one aspect, the hydrolases of the invention are versatilebiocatalysts in organic synthesis, e.g., in the structured synthesis ofoils, lipids and the like. Enzymes of the invention (including, e.g.,esterases such as carboxyl esterases and lipases) can accept a broadrange of substrates, including secondary and tertiary alcohols, e.g.,from a natural product such as alpha-terpineol, linalool and the like.In some aspects, the hydrolases of the invention have good to excellentenantiospecificity (e.g., stereospecificity).

In one aspect, the hydrolase of the invention comprises a GGGX motif. Asdiscussed above, in one aspect, the invention provides a fragment orsubsequence of an enzyme of the invention comprising a catalytic domain(“CD”) or “active site.” In one aspect, a catalytic domain (“CD”) or“active site” comprising peptide, catalytic antibody or polypeptide ofthe invention comprises a GGGX motif. In one aspect, this motif islocated on a protein loop near the binding site of the substrate ester'scarboxylic group. In one aspect, the GGGX motif is involved in theformation of an “oxyanion hole” which stabilizes the anionic carbonyloxygen of a tetrahedral intermediate during the catalytic cycle of esterhydrolysis. In one aspect, the invention provides an esterase or alipase comprising a GGGX motif for the hydrolysis of a tertiary alcoholester. In one aspect, the invention provides an esterase or a lipase forthe hydrolysis of a terpinyl-, linalyl, 2-phenyl-3-butin-2-yl acetateand/or a 3-methyl-1-pentin-3-yl-acetate, wherein the enzyme of theinvention comprises a GGGX motif.

In one aspect, the invention provides an oil (e.g., vegetable oils,cocoa butters, and the like) conversion process comprising at least oneenzyme (e.g., a lipase) of the invention. In one aspect, an oilconversion process comprises a controlled hydrolysis and acylation,e.g., a glycerol acylation, which can result in high purity and a broadend of products. In one aspect, hydrolases (e.g., lipases) of theinvention are used to produce diacylglycerol oils and structurednutritional oils. In one aspect, the invention provides processes forthe esterification of propylene glycol using an enzyme of the invention,e.g., a regio- and/or chemo-selective lipase for mono-substitutedesterification at the sn-1 position. In one aspect, the inventionprovides processes for the structured synthesis of oils with targetedsaturated or unsaturated fatty acid profiles using an enzyme of theinvention, e.g., a regio- and/or chemo-selective lipase for the removalof a saturated fatty acid, or, for the targeted addition of a fatty acidto a glycerol backbone. In one aspect, the invention provides processesfor modifying saturated fatty acids using an enzyme of the invention,e.g., by adding double bonds using an enzyme with desaturase activity(in one aspect, this process is done in whole cell systems). In oneaspect, the invention provides processes for modifying saturated fattyacids using an enzyme of the invention, e.g., by the removal doublebonds using enzymes with hydrogenation and/or dehydrogenation activity(in one aspect, this process is done in whole cell systems). In oneaspect, the invention provides processes for the total hydrolysis oftriglycerides without trans-isomer formation using an enzyme of theinvention, e.g., a non-selective lipase of the invention for totalhydrolysis without formation of trans-isomers. In one aspect, theinvention provides processes for enzyme catalyzed monoesterification ofa glycol, e.g., a propylene glycol, using a hydrolase (e.g., a lipase,an esterase) of the invention. In one aspect, oleic, linoleic oralpha-linolenic acids are used in the enzyme catalyzedmonoesterification. Any oil, e.g., a vegetable oil such as soy, cotton,corn, rice bran or sunflower can be used in this process. The enzyme canbe chemoselective and/or enantioselective. For example, in one aspect, achemoselective enzyme of the invention can be selective for a singleacid, e.g., oleic, linoleic or alpha-linolenic acid individually, or,can be selective for two acids only, e.g., oleic or linoleic acids only,or, linoleic or alpha-linolenic only, etc. Alternatively, an enzyme ofthe invention can be enantioselective (in esterification or hydrolysis).For example, an enzyme can be selective for only a single position, or,selective for only two positions, e.g., only 1,2 esterification, or,only 1,3 esterification, or, only 2,3 esterification (or, in the reversereaction, hydrolysis).

In one aspect, the invention provides processes for the selectiveremoval of fatty acids (e.g., undesirable fatty acids) from oils, e.g.,separating saturated and/or unsaturated fatty acids from oils, using ahydrolase (e.g., a lipase, an esterase) of the invention. The process ofthe invention can separate saturated and/or unsaturated fatty acids fromany oil, e.g., a soy oil. The enzyme can be chemoselective and/orenantioselective. The process can comprise selective acylation with cisisomers, Sn-2 esterification, enzymatic hydrogenation. In one aspect,these processes generate high stability fats and oils, e.g., “healthy”frying oils. The process of the invention can be used to generate oilswith less sulfur, e.g., using a process comprising sulfur removal fromcrude oil. The enzymes of the invention can also be used ininteresterification processes for these and other purposes.

In one aspect, an enzyme of the invention is used to generate a“no-trans” fat oil. In one aspect, a “no-trans” oil is generated from apartially hydrogenated oil to produce a cis-only oil. The enzyme can bechemoselective and/or enantioselective.

In one aspect, the invention provides processes for modifying cocoabutters using an enzyme of the invention. About 80% of cocoa butterscomprise POP, SOS and POS triglycerides (P is palmitic fatty acid, O isoleic fatty acid, S is stearic fatty acid). Thesaturated-unsaturated-saturated fatty acid structure of cocoa buttersimparts their characteristic melting profiles, e.g., in chocolates. Inone aspect, the structured and direct synthetic processes of theinvention are used on cocoa butters to reduce cocoa butter variations orto produce synthetic cocoa butters (“cocoa butter alternatives”). In oneaspect, a chemoselective and/or enantioselective (e.g., aregio-selective) hydrolase (e.g., lipase or esterase) of the inventionis used to make a cocoa butter alternative, e.g., a cocoa buttersubstitute, a cocoa butter replacer and/or a cocoa butter equivalent.Thus, the invention also provides cocoa butter alternatives, includingcocoa butter substitutes, cocoa butter replacers and cocoa butterequivalents and their manufacturing intermediates comprising an enzymeof the invention. A process of the invention (using an enzyme of theinvention) for making cocoa butter alternatives can comprise blending avegetable oil, e.g., a palm oil, with shea or equivalent, illipe orequivalent and Sal sterins or equivalent. In one aspect, the process ofthe invention comprises use of interesterification. The process of theinvention can generate compositional or crystalline forms that mimic“natural” cocoa butter.

In one aspect, the invention provides processes (using an enzyme of theinvention) for producing a diacylglycerol (DAG), e.g., 1, 3diacylglycerol, using a vegetable oil, e.g., a low cost oil. The enzymecan be chemoselective and/or enantioselective. The process of theinvention can result in a DAG-comprising composition having goodstability, long shelf life and high temperature performance.

Enzymatic Processing of Oilseeds

The invention provides compositions (e.g., hydrolase enzymes of theinvention, such as lipases, phospholipases, esterases, proteases) andmethods for enzymatic processing of oilseeds, including soybean, canola,coconut, avocado and olive paste. In one aspect, these processes of theinvention can increase the oil yield and to improve the nutritionalquality of the obtained meals. In some aspects, enzymatic processing ofoilseeds using compositions and methods of the invention will provideeconomical and environmental benefits, as well as alternativetechnologies for oil extraction and processing food for human and animalconsumption. In alternative aspects, the processes of the inventioncomprise use of any hydrolase of the invention, e.g., a phospholipasesof the invention (or another phospholipase), a protease of the invention(or another protease), phosphatases, phytases, xylanases, an amylase,e.g., α-amylases, a glucanase, e.g., β-glucanases, a polygalacturonase,galactolipases, a cellulase, a hemicellulase, a pectinases and/or otherplant cell wall degrading enzymes, as well as mixed enzyme preparationsand cell lysates, or enzyme preparations from recombinant sources, e.g.,host cells or transgenic plants.

In alternative aspects, the processes of the invention can be practicedin conjunction with other processes, e.g., enzymatic treatments, e.g.,with carbohydrases, including cellulase, hemicellulase and other sidedegrading activities, or, chemical processes, e.g., hexane extraction ofsoybean oil. The enzymatic treatment can increase the oil extractabilityby 8-10% when the enzymatic treatment is carried out prior to thesolvent extraction.

In alternative aspects, the processes of the invention can be practicedwith aqueous extraction processes. The aqueous extraction methods can beenvironmentally cleaner alternative technologies for oil extraction. Lowextraction yields of aqueous process can be overcome by using enzymesthat hydrolyze the structural polysaccharides forming the cell wall ofoilseeds, or that hydrolyze the proteins which form the cell and lipidbody membranes, e.g., utilizing digestions comprising cellulase,hemicellulase, and/or protopectinase for extraction of oil from soybeancells. In one aspect, methods are practiced with an enzyme of theinvention as described by Kasai (2003) J. Agric. Food Chem.51:6217-6222, who reported that the most effective enzyme to digest thecell wall was cellulase.

In one aspect, proteases of the invention or other proteases are used incombination with the methods of the invention. The combined effect ofoperational variables and enzyme activity of a protease and cellulase onoil and protein extraction yields combined with other processparameters, such as enzyme concentration, time of hydrolysis, particlesize and solid-to-liquid ratio has been evaluated. In one aspect,methods are practiced with an enzyme of the invention as described byRosenthal (2001) Enzyme and Microb. Tech. 28:499-509, who reported thatuse of protease can result in significantly higher yields of oil andprotein over the control when heat treated flour is used.

In one aspect, complete protein, pectin, and hemicellulose extractionare used in combination with the methods of the invention. The plantcell consists of a series of polysaccharides often associated with orreplaced by proteins or phenolic compounds. Most of these carbohydratesare only partially digested or poorly utilized by the digestive enzymes.The disruption of these structures through processing or degradingenzymes can improve their nutrient availability. In one aspect, methodsare practiced with an enzyme of the invention as described by Ouhida(2002) J. Agric. Food Chem. 50:1933-1938, who reported that asignificant degradation of the soybean cell wall cellulose (up to 20%)has been achieved after complete protein, pectin, and hemicelluloseextraction.

In one aspect, the methods of the invention further compriseincorporation of various enzymatic treatments in the treatment of seeds,e.g., canola seeds, these treatments comprising use of proteases of theinvention (or other proteases), cellulases, and hemicellulases (invarious combinations with each other and with one or more enzymes of theinvention). For example, the methods can comprise enzymatic treatmentsof canola seeds at 20 to 40 moisture during the incubation with enzymesprior to a conventional process; as described, e.g., by Sosulski (1990)Proc. Can. Inst. Food Sci. Technol. 3:656. The methods of the inventioncan further comprise incorporation of proteases of the invention (orother proteases), α-amylases, polygalacturonases (in variouscombinations with each other and with one or more enzymes of theinvention) to hydrolyze cellular material in coconut meal and releasethe coconut oil, which can be recovered by centrifugation, as described,e.g., by McGlone (1986) J. of Food Sci. 51:695-697. The methods of theinvention can further comprise incorporation of pectinases, α-amylases,proteases of the invention (or other proteases), cellulases in differentcombinations (with each other and with one or more enzymes of theinvention) to result in significant yield improvement (˜70% in the bestcase) during enzymatic extraction of avocado oil, as described, e.g., byBuenrostro (1986) Biotech. Letters 8(7):505-506. In processes of theinvention for olive oil extraction, olive paste is treated withcellulase, hemicellulase, poligalacturonase, pectin-methyltransferase,protease of the invention (or other proteases) and their combinations(with each other and with one or more enzymes of the invention), asdescribed, e.g., by Montedoro (1976) Acta Vitamin. Enzymol. (Milano)30:13.

Oil Degumming and Vegetable Oil Processing

The enzymes of the invention (e.g., lipases, phospholipases, esterases,proteases of the invention) can be used in various vegetable oilprocessing steps, such as in vegetable oil extraction, particularly, inthe removal of “phospholipid gums” in a process called “oil degumming,”.

In one aspect, the invention provides oil degumming processes comprisinguse of a hydrolase of the invention having a phospholipase C (PLC)activity. In one aspect, the process further comprises addition of a PLAof the invention and/or a patatin-like phospholipase of the invention.In one aspect, all enzymes are added together, or, alternatively, thePLC addition is followed by PLA and/or patatin addition. In one aspect,this process provides a yield improvement as a result of the PLCtreatment. In one aspect, this process provides an additional decreaseof the phosphorus content of the oil as a result of the PLA treatment.

The invention provides methods for processing vegetable oils fromvarious sources, such as rice bran, soybeans, rapeseed, peanuts andother nuts, sesame, sunflower, palm and corn. The methods can used inconjunction with processes based on extraction with as hexane, withsubsequent refining of the crude extracts to edible oils, including useof the methods and enzymes of the invention. The first step in therefining sequence is the so-called “degumming” process, which serves toseparate phosphatides by the addition of water. The materialprecipitated by degumming is separated and further processed to mixturesof lecithins. The commercial lecithins, such as soybean lecithin andsunflower lecithin, are semi-solid or very viscous materials. Theyconsist of a mixture of polar lipids, mainly phospholipids, and oil,mainly triglycerides.

The enzymes (e.g., phospholipases) of the invention can be used in any“degumming” procedure, including water degumming, ALCON oil degumming(e.g., for soybeans), safinco degumming, “super degumming,” UFdegumming, TOP degumming, uni-degumming, dry degumming and ENZYMAX™degumming See, e.g., U.S. Pat. Nos. 6,355,693; 6,162,623; 6,103,505;6,001,640; 5,558,781; 5,264,367. Various “degumming” proceduresincorporated by the methods of the invention are described in Bockisch,M. (1998) In Fats and Oils Handbook, The extraction of Vegetable Oils(Chapter 5), 345-445, AOCS Press, Champaign, Ill. The enzymes (e.g.,phospholipases) of the invention can be used in the industrialapplication of enzymatic degumming of triglyceride oils as described,e.g., in EP 513 709.

In one aspect, hydrolases (e.g., phospholipases) of the invention areused to treat vegetable oils, e.g., crude oils, such as rice bran, soy,canola, flower and the like. In one aspect, this improves the efficiencyof the degumming process. In one aspect, the invention provides methodsfor enzymatic degumming under conditions of low water, e.g., in therange of between about 0.1% to 20% water, or, 0.5% to 10% water. In oneaspect, this results in the improved separation of a heavy phase fromthe oil phase during centrifugation. The improved separation of thesephases can result in more efficient removal of phospholipids from theoil, including both hydratable and nonhydratable oils. In one aspect,this can produce a gum fraction that contains less entrained neutraloil, thereby improving the overall yield of oil during the degummingprocess.

The hydrolases (e.g., phospholipases) of the invention can be used inthe industrial application of enzymatic degumming as described, e.g., inCA 1102795, which describes a method of isolating polar lipids fromcereal lipids by the addition of at least 50% by weight of water. Thismethod is a modified degumming in the sense that it utilizes theprinciple of adding water to a crude oil mixture.

In one aspect, the invention provides enzymatic processes comprising useof phospholipases of the invention (e.g., a PLC) comprising hydrolysisof hydrated phospholipids in oil at a temperature of about 20° C. to 40°C., at an alkaline pH, e.g., a pH of about pH 8 to pH 10, using areaction time of about 3 to 10 minutes. This can result in less than 10ppm final oil phosphorus levels. The invention also provides enzymaticprocesses comprising use of phospholipases of the invention (e.g., aPLC) comprising hydrolysis of hydratable and non-hydratablephospholipids in oil at a temperature of about 50° C. to 60° C., at a pHslightly below neutral, e.g., of about pH 5 to pH 6.5, using a reactiontime of about 30 to 60 minutes. This can result in less than 10 ppmfinal oil phosphorus levels.

In one aspect, the invention provides enzymatic processes that utilize aphospholipase C enzyme to hydrolyze a glyceryl phosphoester bond andthereby enable the return of the diacylglyceride portion ofphospholipids back to the oil, e.g., a vegetable, fish or algae oil (a“phospholipase C (PLC) caustic refining aid”); and, reduce thephospholipid content in a degumming step to levels low enough for highphosphorous oils to be physically refined (a “phospholipase C (PLC)degumming aid”). The two approaches can generate different values andhave different target applications.

In various exemplary processes of the invention, a number of distinctsteps compose the degumming process preceding the core bleaching anddeodorization refining processes. These steps include heating, mixing,holding, separating and drying. Following the heating step, water andoften acid are added and mixed to allow the insoluble phospholipid “gum”to agglomerate into particles which may be separated. While waterseparates many of the phosphatides in degumming, portions of thephospholipids are non-hydratable phosphatides (NHPs) present as calciumor magnesium salts. Degumming processes address these NHPs by theaddition of acid. Following the hydration of phospholipids, the oil ismixed, held and separated by centrifugation. Finally, the oil is driedand stored, shipped or refined. The resulting gums are either processedfurther for lecithin products or added back into the meal.

In various exemplary processes of the invention phosphorous levels arereduced low enough for physical refining. The separation process canresult in potentially higher yield losses than caustic refining.Additionally, degumming processes may generate waste products that maynot be sold as commercial lecithin. Therefore, these processes have notachieved a significant share of the market and caustic refiningprocesses continue to dominate the industry for rice bran, soy, canolaand sunflower. Note however, that a phospholipase C enzyme employed in aspecial degumming process would decrease gum formation and return thediglyceride portion of the phospholipid back to the oil.

In one aspect, a phospholipase C enzyme of the invention hydrolyzes aphosphatide at a glyceryl phosphoester bond to generate a diglycerideand water-soluble phosphate compound. The hydrolyzed phosphatide movesto the aqueous phase, leaving the diglyceride in the oil phase. Oneobjective of the PLC “Caustic Refining Aid” is to convert thephospholipid gums formed during neutralization into a diacylglyceridethat will migrate back into the oil phase. In contrast, one objective ofthe “PLC Degumming Aid” is to reduce the phospholipids in crude oil to aphosphorous equivalent of less than 10 parts per million.

In one aspect, a phospholipase C enzyme of the invention will hydrolyzethe phosphatide from both hydratable and non-hydratable phospholipids inneutralized crude and degummed oils before bleaching and deodorizing.The target enzyme can be applied as a drop-in product in the existingcaustic neutralization process. In this aspect, the enzyme will not berequired to withstand extreme pH levels if it is added after theaddition of caustic.

In one aspect, a phospholipase of the invention enables phosphorous tobe removed to the low levels acceptable in physical refining. In oneaspect, a PLC of the invention will hydrolyze the phosphatide from bothhydratable and non-hydratable phospholipids in crude oils beforebleaching and deodorizing. The target enzyme can be applied as a drop-inproduct in the existing degumming operation. Given sub-optimal mixing incommercial equipment, it is likely that acid will be required to bringthe non-hydratable phospholipids in contact with the enzyme at theoil/water interface. Therefore, in one aspect, an acid-stable PLC of theinvention is used.

In one aspect, a PLC Degumming Aid process of the invention caneliminate losses in one, or all three, areas: 1) Oil lost in gumformation & separation; 2) Saponified oil in caustic addition; 3) Oiltrapped in clay in bleaching. Losses associated in a PLC process can beestimated to be about 0.8% versus 5.2% on a mass basis due to removal ofthe phosphatide. Additional potential benefits of this process of theinvention include the following:

-   -   Reduced adsorbents—less adsorbents required with lower (<5 ppm)        phosphorous    -   Lower chemical usage—less chemical and processing costs        associated with hydration of non-hydratable phospholipids    -   Lower waste generation—less water required to remove phosphorous        from oil

Oils processed (e.g., “degummed”) by the methods of the inventioninclude plant oilseeds, e.g., rice bran, soybean oil, rapeseed oil andsunflower oil. In one aspect, the “PLC Caustic Refining Aid” of theinvention can save 1.2% over existing caustic refining processes. Therefining aid application addresses soy oil that has been degummed forlecithin and these are also excluded from the value/load calculations.

Other processes that can be used with a phospholipase of the invention,e.g., a phospholipase A₁ of the invention can convert non-hydratablenative phospholipids to a hydratable form. In one aspect, the enzyme issensitive to heat. This may be desirable, since heating the oil candestroy the enzyme. However, the degumming reaction must be adjusted topH 4-5 and 60° C. to accommodate this enzyme. At 300 Units/kg oilsaturation dosage, this exemplary process is successful at takingpreviously water-degummed oil phosphorous content down to ≧10 ppm P.Advantages can be decreased H₂O content and resultant savings in usage,handling and waste.

In addition to these various “degumming” processes, the enzymes of theinvention can be used in any vegetable oil processing step. For example,phospholipase enzymes of the invention can be used in place of PLA,e.g., phospholipase A2, in any vegetable oil processing step. Oils thatare “processed” or “degummed” in the methods of the invention includesoybean oils, rapeseed oils, corn oils, oil from rice bran oils, palmkernels, canola oils, sunflower oils, sesame oils, peanut oils, and thelike. The main products from this process include triglycerides.

In one exemplary process, when the enzyme is added to and reacted with acrude oil, the amount of phospholipase employed is about 10-10,000units, or, alternatively, about, 100-2,000 units, per 1 kg of crude oil.The enzyme treatment is conducted for 5 min to 10 hours at a temperatureof 30° C. to 90° C., or, alternatively, about, 40° C. to 70° C. Theconditions may vary depending on the optimum temperature of the enzyme.The amount of water added to dissolve the enzyme is 5-1,000 wt. partsper 100 wt. parts of crude oil, or, alternatively, about, 10 to 200 wt.parts per 100 wt. parts of crude oil.

Upon completion of such enzyme treatment, the enzyme liquid is separatedwith an appropriate means such as a centrifugal separator and theprocessed oil is obtained. Phosphorus-containing compounds produced byenzyme decomposition of gummy substances in such a process arepractically all transferred into the aqueous phase and removed from theoil phase. Upon completion of the enzyme treatment, if necessary, theprocessed oil can be additionally washed with water or organic orinorganic acid such as, e.g., acetic acid, phosphoric acid, succinicacid, and the like, or with salt solutions.

In one exemplary process for ultra-filtration degumming, the enzyme isbound to a filter or the enzyme is added to an oil prior to filtrationor the enzyme is used to periodically clean filters.

In one aspect, the invention provides processes using a hydrolase of theinvention, e.g., a phospholipase of the invention, such as aphospholipase-specific phosphohydrolase of the invention, or anotherphospholipase, in a modified “organic refining process,” which cancomprise addition of the enzyme (e.g., a hydrolase, such as a PLC) in acitric acid holding tank.

Enzymes of the invention are used to improve oil extraction and oildegumming (e.g., vegetable oils). In one aspect, a hydrolase (e.g.,phospholipase, such as a PLC) of the invention and at least one plantcell wall degrader (e.g., a cellulase, a hemicellulase or the like, tosoften walls and increase yield at extraction) is used in a process ofthe invention. In this exemplary approach to using enzymes of theinvention to improve oil extraction and oil degumming, a hydrolase(e.g., phospholipase C) of the invention as well as other hydrolases(e.g., a cellulase, a hemicellulase, an esterase of the invention oranother esterase, a protease of the invention of the invention oranother protease and/or a phosphatase) are used during the crushingsteps associated with oil production (including but not limited tosoybean, canola, rice bran and sunflower oil). By using enzymes prior toor in place of solvent extraction, it is possible to increase oil yieldand reduce the amount of hydratable and non-hydratable phospholipids inthe crude oil. The reduction in non-hydratable phospholipids may resultfrom conversion of potentially non-hydratable phospholipids todiacylglycerol and corresponding phosphate-ester prior to complexationwith calcium or magnesium. The overall reduction of phospholipids in thecrude oil will result in improved yields during refining with thepotential for eliminating the requirement for a separate degumming stepprior to bleaching and deodorization.

In one exemplary process for a phospholipase-mediated physical refiningaid, water and enzyme are added to crude oil. In one aspect, a PLC or aPLD and a phosphatase are used in the process. In phospholipase-mediatedphysical refining, the water level can be low, i.e. 0.5-5% and theprocess time should be short (less than 2 hours, or, less than 60minutes, or, less than 30 minutes, or, less than 15 minutes, or, lessthan 5 minutes). The process can be run at different temperatures (25°C. to 70° C.), using different acids and/or caustics, at different pHs(e.g., 3-10).

In alternate aspects, water degumming is performed first to collectlecithin by centrifugation and then PLC or PLC and PLA is added toremove non-hydratable phospholipids (the process should be performedunder low water concentration). In another aspect, water degumming ofcrude oil to less than 10 ppm (edible oils) and subsequent physicalrefining (less than 50 ppm for biodiesel) is performed. In one aspect,an emulsifier is added and/or the crude oil is subjected to an intensemixer to promote mixing. Alternatively, an emulsion-breaker is addedand/or the crude oil is heated to promote separation of the aqueousphase. In another aspect, an acid is added to promote hydration ofnon-hydratable phospholipids. Additionally, phospholipases can be usedto mediate purification of phytosterols from the gum/soapstock.

The enzymes of the invention can be used in any oil processing method,e.g., degumming or equivalent processes. For example, the enzymes of theinvention can be used in processes as described in U.S. Pat. Nos.5,558,781; 5,264,367; 6,001,640. The process described in U.S. Pat. No.5,558,781 uses either phospholipase A1, A2 or B, essentially breakingdown lecithin in the oil that behaves as an emulsifier.

The enzymes and methods of the invention can be used in processes forthe reduction of phosphorus-containing components in edible oilscomprising a high amount of non-hydratable phosphorus by using of aphospholipase of the invention, e.g., a polypeptide having aphospholipase A and/or B activity, as described, e.g., in EP PatentNumber: EP 0869167. In one aspect, the edible oil is a crude oil, aso-called “non-degummed oil.” In one aspect, the method treat anon-degummed oil, including pressed oils or extracted oils, or a mixturethereof, from, e.g., rice bran, rapeseed, soybean, sesame, peanut, cornor sunflower. The phosphatide content in a crude oil can vary from 0.5to 3% w/w corresponding to a phosphorus content in the range of 200 to1200 ppm, or, in the range of 250 to 1200 ppm. Apart from thephosphatides, the crude oil can also contains small concentrations ofcarbohydrates, sugar compounds and metal/phosphatide acid complexes ofCa, Mg and Fe. In one aspect, the process comprises treatment of aphospholipid or lysophospholipid with the phospholipase of the inventionso as to hydrolyze fatty acyl groups. In one aspect, the phospholipid orlysophospholipid comprises lecithin or lysolecithin. In one aspect ofthe process the edible oil has a phosphorus content from between about50 to 250 ppm, and the process comprises treating the oil with aphospholipase of the invention so as to hydrolyze a major part of thephospholipid and separating an aqueous phase containing the hydrolyzedphospholipid from the oil. In one aspect, prior to the enzymaticdegumming process the oil is water-degummed In one aspect, the methodsprovide for the production of an animal feed comprising mixing thephospholipase of the invention with feed substances and at least onephospholipid.

The enzymes and methods of the invention can be used in processes of oildegumming as described, e.g., in WO 98/18912. The phospholipases of theinvention can be used to reduce the content of phospholipid in an edibleoil. The process can comprise treating the oil with a phospholipase ofthe invention to hydrolyze a major part of the phospholipid andseparating an aqueous phase containing the hydrolyzed phospholipid fromthe oil. This process is applicable to the purification of any edibleoil, which contains a phospholipid, e.g. vegetable oils, such as ricebran, soybean oil, rapeseed oil and sunflower oil, fish oils, algae andanimal oils and the like. Prior to the enzymatic treatment, thevegetable oil is preferably pretreated to remove slime (mucilage), e.g.by wet refining. The oil can contain 50-250 ppm of phosphorus asphospholipid at the start of the treatment with phospholipase, and theprocess of the invention can reduce this value to below 5-10 ppm.

The enzymes of the invention can be used in processes as described in JPApplication No.: H5-132283, filed Apr. 25, 1993, which comprises aprocess for the purification of oils and fats comprising a step ofconverting phospholipids present in the oils and fats into water-solublesubstances containing phosphoric acid groups and removing them aswater-soluble substances. An enzyme action is used for the conversioninto water-soluble substances. An enzyme having a phospholipase Cactivity is preferably used as the enzyme.

The enzymes of the invention can be used in processes as described asthe “Organic Refining Process,” (ORP) (IPH, Omaha, Nebr.) which is amethod of refining seed oils. ORP may have advantages over traditionalchemical refining, including improved refined oil yield, value addedco-products, reduced capital costs and lower environmental costs.

The enzymes of the invention can be used in processes for the treatmentof an oil or fat, animal or vegetal, raw, semi-processed or refined,comprising adding to such oil or fat at least one enzyme of theinvention that allows hydrolyzing and/or depolymerizing thenon-glyceridic compounds contained in the oil, as described, e.g., in EPApplication number: 82870032.8. Exemplary methods of the invention forhydrolysis and/or depolymerization of non-glyceridic compounds in oilsare:

-   1) The addition and mixture in oils and fats of an enzyme of the    invention or enzyme complexes previously dissolved in a small    quantity of appropriate solvent (for example water). A certain    number of solvents are possible, but a non-toxic and suitable    solvent for the enzyme is chosen. This addition may be done in    processes with successive loads, as well as in continuous processes.    The quantity of enzyme(s) necessary to be added to oils and fats,    according to this process, may range, depending on the enzymes and    the products to be processed, from 20 to 400 ppm, i.e., from 0.02 kg    to 0.4 kg of enzyme for 1000 kg of oil or fat, and preferably from    20 to 100 ppm, i.e., from 0.02 to 0.1 kg of enzyme for 1000 kg of    oil, these values being understood to be for concentrated enzymes,    i.e., without diluent or solvent.-   2) Passage of the oil or fat through a fixed or insoluble filtering    bed of enzyme(s) of the invention on solid or semi-solid supports,    preferably presenting a porous or fibrous structure. In this    technique, the enzymes are trapped in the micro-cavities of the    porous or fibrous structure of the supports. These consist, for    example, of resins or synthetic polymers, cellulose carbonates, gels    such as agarose, filaments of polymers or copolymers with porous    structure, trapping small droplets of enzyme in solution in their    cavities. Concerning the enzyme concentration, it is possible to go    up to the saturation of the supports.-   3) Dispersion of the oils and fats in the form of fine droplets, in    a diluted enzymatic solution, preferably containing 0.2 to 4% in    volume of an enzyme of the invention. This technique is described,    e.g., in Belgian patent No. 595,219. A cylindrical column with a    height of several meters, with conical lid, is filled with a diluted    enzymatic solution. For this purpose, a solvent that is non-toxic    and non-miscible in the oil or fat to be processed, preferably    water, is chosen. The bottom of the column is equipped with a    distribution system in which the oil or fat is continuously injected    in an extremely divided form (approximately 10,000 flux per m²).    Thus an infinite number of droplets of oil or fat are formed, which    slowly rise in the solution of enzymes and meet at the surface, to    be evacuated continuously at the top of the conical lid of the    reactor.

Palm oil can be pre-treated before treatment with an enzyme of theinvention. For example, about 30 kg of raw palm oil is heated to +50° C.1% solutions were prepared in distilled water with cellulases andpectinases. 600 g of each of these was added to aqueous solutions of theoil under strong agitation for a few minutes. The oil is then kept at+50° C. under moderate agitation, for a total reaction time of twohours. Then, temperature is raised to +90° C. to deactivate the enzymesand prepare the mixture for filtration and further processing. The oilis dried under vacuum and filtered with a filtering aid.

The enzymes of the invention can be used in processes as described in EPpatent EP 0 513 709 B2. For example, the invention provides a processfor the reduction of the content process for the reduction of thecontent of phosphorus-containing components in animal and vegetable oilsby enzymatic decomposition using a phospholipase of the invention. Apredemucilaginated animal and vegetable oil with a phosphorus content of50 to 250 ppm is agitated with an organic carboxylic acid and the pHvalue of the resulting mixture set to pH 4 to pH 6, an enzyme solutionwhich contains phospholipase A₁, A₂, or B of the invention is added tothe mixture in a mixing vessel under turbulent stirring and with theformation of fine droplets, where an emulsion with 0.5 to 5% by weightrelative to the oil is formed, said emulsion being conducted through atleast one subsequent reaction vessel under turbulent motion during areaction time of 0.1 to 10 hours at temperatures in the range of 20 to80° C. and where the treated oil, after separation of the aqueoussolution, has a phosphorus content under 5 ppm.

The organic refining process is applicable to both crude and degummedoil. The process uses inline addition of an organic acid undercontrolled process conditions, in conjunction with conventionalcentrifugal separation. The water separated naturally from the vegetableoil phospholipids (“VOP”) is recycled and reused. The total water usagecan be substantially reduced as a result of the Organic RefiningProcess.

The phospholipases and methods of the invention can also be used in theenzymatic treatment of edible oils, as described, e.g., in U.S. Pat. No.6,162,623. In this exemplary methods, the invention provides anamphiphilic enzyme. It can be immobilized, e.g., by preparing anemulsion containing a continuous hydrophobic phase and a dispersedaqueous phase containing the enzyme and a carrier for the enzyme andremoving water from the dispersed phase until this phase turns intosolid enzyme coated particles. The enzyme can be a lipase. Theimmobilized lipase can be used for reactions catalyzed by lipase such asinteresterification of mono-, di- or triglycerides, de-acidification ofa triglyceride oil, or removal of phospholipids from a triglyceride oilwhen the lipase is a phospholipase. The aqueous phase may contain afermentation liquid, an edible triglyceride oil may be the hydrophobicphase, and carriers include sugars, starch, dextran, water solublecellulose derivatives and fermentation residues. This exemplary methodcan be used to process triglycerides, diglycerides, monoglycerides,glycerol, phospholipids or fatty acids, which may be in the hydrophobicphase. In one aspect, the process for the removal of phospholipids fromtriglyceride oil comprising mixing a triglyceride oil containingphospholipids with a preparation containing a phospholipase of theinvention; hydrolyzing the phospholipids to lysophospholipid; separatingthe hydrolyzed phospholipids from the oil, wherein the phospholipase isan immobilized phospholipase.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used in the enzymatictreatment of edible oils, as described, e.g., in U.S. Pat. No.6,127,137. One exemplary method hydrolyzes both fatty acyl groups inintact phospholipid. A phospholipase of the invention used in thismethod can have no lipase activity and can be active at very low pH.These properties make it very suitable for use in oil degumming, asenzymatic and alkaline hydrolysis (saponification) of the oil can bothbe suppressed.

In one aspect, the invention provides a process for hydrolyzing fattyacyl groups in a phospholipid or lysophospholipid comprising treatingthe phospholipid or lysophospholipid with the phospholipase thathydrolyzes both fatty acyl groups in a phospholipid and is essentiallyfree of lipase activity. In one aspect, the phospholipase of theinvention has a temperature optimum at about 50° C., measured at pH 3 topH 4 for 10 minutes, and a pH optimum of about pH 3, measured at 40° C.for about 10 minutes. In one aspect, the phospholipid orlysophospholipid comprises lecithin or lysolecithin. In one aspect,after hydrolyzing a major part of the phospholipid, an aqueous phasecontaining the hydrolyzed phospholipid is separated from the oil. In oneaspect, the invention provides a process for removing phospholipid froman edible oil, comprising treating the oil at pH 1.5 to 3 with adispersion of an aqueous solution of the phospholipase of the invention,and separating an aqueous phase containing the hydrolyzed phospholipidfrom the oil. In one aspect, the oil is treated to remove mucilage priorto the treatment with the phospholipase. In one aspect, the oil prior tothe treatment with the phospholipase contains the phospholipid in anamount corresponding to 50 to 250 ppm of phosphorus. In one aspect, thetreatment with phospholipase is done at 30° C. to 45° C. for 1 to 12hours at a phospholipase dosage of 0.1 to 10 mg/l in the presence of 0.5to 5% of water.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used in the enzymatictreatment of edible oils, as described, e.g., in U.S. Pat. No.6,025,171. In this exemplary method, enzymes of the invention areimmobilized by preparing an emulsion containing a continuous hydrophobicphase, such as a triglyceride oil, and a dispersed aqueous phasecontaining an amphiphilic enzyme, such as lipase or a phospholipase ofthe invention, and carrier material that is partly dissolved and partlyundissolved in the aqueous phase, and removing water from the aqueousphase until the phase turns into solid enzyme coated carrier particles.The undissolved part of the carrier material may be a material that isinsoluble in water and oil, or a water soluble material in undissolvedform because the aqueous phase is already saturated with the watersoluble material. The aqueous phase may be formed with a crude lipasefermentation liquid containing fermentation residues and biomass thatcan serve as carrier materials Immobilized lipase is useful for esterre-arrangement and de-acidification in oils. After a reaction, theimmobilized enzyme can be regenerated for a subsequent reaction byadding water to obtain partial dissolution of the carrier, and with theresultant enzyme and carrier-containing aqueous phase dispersed in ahydrophobic phase evaporating water to again form enzyme coated carrierparticles.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used in the enzymatictreatment of edible oils, as described, e.g., in U.S. Pat. No.6,143,545. This exemplary method is used for reducing the content ofphosphorous containing components in an edible oil comprising a highamount of non-hydratable phosphorus content using a phospholipase of theinvention. In one aspect, the method is used to reduce the content ofphosphorus containing components in an edible oil having anon-hydratable phosphorus content of at least 50 ppm measured bypre-treating the edible oil, at 60° C., by addition of a solutioncomprising citric acid monohydrate in water (added water vs. oil equals4.8% w/w; (citric acid) in water phase=106 mM, in water/oil emulsion=4.6mM) for 30 minutes; transferring 10 ml of the pre-treated water in oilemulsion to a tube; heating the emulsion in a boiling water bath for 30minutes; centrifuging at 5000 rpm for 10 minutes, transferring about 8ml of the upper (oil) phase to a new tube and leaving it to settle for24 hours; and drawing 2 g from the upper clear phase for measurement ofthe non-hydratable phosphorus content (ppm) in the edible oil. Themethod also can comprise contacting an oil at a pH from about pH 5 to 8with an aqueous solution of a phospholipase A or B of the invention(e.g., PLA1, PLA2, or a PLB), which solution is emulsified in the oiluntil the phosphorus content of the oil is reduced to less than 11 ppm,and then separating the aqueous phase from the treated oil.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used in the enzymatictreatment of edible oils, as described, e.g., in U.S. Pat. No.5,532,163. The invention provides processes for the refining of oil andfat by which phospholipids in the oil and fat to be treated can bedecomposed and removed efficiently. In one aspect, the inventionprovides a process for the refining of oil and fat which comprisesreacting, in an emulsion, the oil and fat with an enzyme of theinvention, e.g., an enzyme having an activity to decomposeglycerol-fatty acid ester bonds in glycerophospholipids (e.g., a PLA2 ofthe invention); and another process in which the enzyme-treated oil andfat is washed with water or an acidic aqueous solution. In one aspect,the acidic aqueous solution to be used in the washing step is a solutionof at least one acid, e.g., citric acid, acetic acid, phosphoric acidand salts thereof. In one aspect, the emulsified condition is formedusing 30 weight parts or more of water per 100 weight parts of the oiland fat. Since oil and fat can be purified without employing theconventional alkali refining step, generation of washing waste water andindustrial waste can be reduced. In addition, the recovery yield of oilis improved because loss of neutral oil and fat due to their inclusionin these wastes does not occur in the inventive process. In one aspect,the invention provides a process for refining oil and fat containingabout 100 to 10,000 ppm of phospholipids which comprises: reacting, inan emulsified condition, said oil and fat with an enzyme of theinvention having activity to decompose glycerol-fatty acid ester bondsin glycerophospholipids. In one aspect, the invention provides processesfor refining oil and fat containing about 100 to 10,000 ppm ofphospholipids which comprises reacting, in an emulsified condition, oiland fat with an enzyme of the invention having activity to decomposeglycerol-fatty acid ester bonds in glycerophospholipids; andsubsequently washing the treated oil and fat with a washing water.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used in the enzymatictreatment of edible oils, as described, e.g., in U.S. Pat. No.5,264,367. The content of phosphorus-containing components and the ironcontent of an edible vegetable or animal oil, such as an oil, e.g.,soybean oil, which has been wet-refined to remove mucilage, are reducedby enzymatic decomposition by contacting the oil with an aqueoussolution of an enzyme of the invention, e.g., a phospholipase A1, A2, orB, and then separating the aqueous phase from the treated oil. In oneaspect, the invention provides an enzymatic method for decreasing thecontent of phosphorus- and iron-containing components in oils, whichhave been refined to remove mucilage. An oil, which has been refined toremove mucilage, can be treated with an enzyme of the invention, e.g.,phospholipase C, A1, A2, or B. Phosphorus contents below 5 ppm and ironcontents below 1 ppm can be achieved. The low iron content can beadvantageous for the stability of the oil.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used for preparingtransesterified oils, as described, e.g., in U.S. Pat. No. 5,288,619.The invention provides methods for enzymatic transesterification forpreparing a margarine oil having both low trans-acid and lowintermediate chain fatty acid content. The method includes the steps ofproviding a transesterification reaction mixture containing a stearicacid source material and an edible liquid vegetable oil,transesterifying the stearic acid source material and the vegetable oilusing a 1-, 3-positionally specific lipase, and then finallyhydrogenating the fatty acid mixture to provide a recycle stearic acidsource material for a recyclic reaction with the vegetable oil. Theinvention also provides a counter-current method for preparing atransesterified oil. The method includes the steps of providing atransesterification reaction zone containing a 1-, 3-positionallyspecific lipase, introducing a vegetable oil into thetransesterification zone, introducing a stearic acid source material,conducting a supercritical gas or subcritical liquefied gascounter-current fluid, carrying out a transesterification reaction ofthe triglyceride stream with the stearic acid or stearic acid monoesterstream in the reaction zone, withdrawing a transesterified triglyceridemargarine oil stream, withdrawing a counter-current fluid phase,hydrogenating the transesterified stearic acid or stearic acid monoesterto provide a hydrogenated recycle stearic acid source material, andintroducing the hydrogenated recycle stearic acid source material intothe reaction zone.

In one aspect, the highly unsaturated phospholipid compound may beconverted into a triglyceride by appropriate use of a phospholipase C ofthe invention to remove the phosphate group in the sn-3 position,followed by 1,3 lipase acyl ester synthesis. The 2-substitutedphospholipid may be used as a functional food ingredient directly, ormay be subsequently selectively hydrolyzed in reactor 160 using animmobilized phospholipase C of the invention to produce a 1-diglyceride,followed by enzymatic esterification as described herein to produce atriglyceride product having a 2-substituted polyunsaturated fatty acidcomponent.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used in a vegetableoil enzymatic degumming process as described, e.g., in U.S. Pat. No.6,001,640. This method of the invention comprises a degumming step inthe production of edible oils. Vegetable oils from which hydratablephosphatides have been eliminated by a previous aqueous degummingprocess are freed from non-hydratable phosphatides by enzymatictreatment using a phospholipase of the invention. The process can begentle, economical and environment-friendly. Phospholipases that onlyhydrolyze lysolecithin, but not lecithin, are used in this degummingprocess.

In one aspect, to allow the enzyme of the invention to act, both phases,the oil phase and the aqueous phase that contain the enzyme, must beintimately mixed. It may not be sufficient to merely stir them. Gooddispersion of the enzyme in the oil is aided if it is dissolved in asmall amount of water, e.g., 0.5-5 weight-% (relative to the oil), andemulsified in the oil in this form, to form droplets of less than 10micrometers in diameter (weight average). The droplets can be smallerthan 1 micrometer. Turbulent stirring can be done with radial velocitiesabove 100 cm/sec. The oil also can be circulated in the reactor using anexternal rotary pump. The aqueous phase containing the enzyme can alsobe finely dispersed by means of ultrasound action. A dispersionapparatus can be used.

The enzymatic reaction probably takes place at the border surfacebetween the oil phase and the aqueous phase. It is the goal of all thesemeasures for mixing to create the greatest possible surface for theaqueous phase which contains the enzyme. The addition of surfactantsincreases the microdispersion of the aqueous phase. In some cases,therefore, surfactants with HLB values above 9, such as Na-dodecylsulfate, are added to the enzyme solution, as described, e.g., in EP-A 0513 709. A similar effective method for improving emulsification is theaddition of lysolecithin. The amounts added can lie in the range of0.001% to 1%, with reference to the oil. The temperature during enzymetreatment is not critical. Temperatures between 20° C. and 80° C. can beused, but the latter can only be applied for a short time. In thisaspect, a phospholipase of the invention having a good temperatureand/or low pH tolerance is used. Application temperatures of between 30°C. and 50° C. are optimal. The treatment period depends on thetemperature and can be kept shorter with an increasing temperature.Times of 0.1 to 10 hours, or, 1 to 5 hours are generally sufficient. Thereaction takes place in a degumming reactor, which can be divided intostages, as described, e.g., in DE-A 43 39 556. Therefore continuousoperation is possible, along with batch operation. The reaction can becarried out in different temperature stages. For example, incubation cantake place for 3 hours at 40° C., then for 1 hour at 60° C. If thereaction proceeds in stages, this also opens up the possibility ofadjusting different pH values in the individual stages. For example, inthe first stage the pH of the solution can be adjusted to 7, forexample, and in a second stage to 2.5, by adding citric acid. In atleast one stage, however, the pH of the enzyme solution must be below 4,or, below 3. If the pH was subsequently adjusted below this level, adeterioration of effect may be found. Therefore the citric acid can beadded to the enzyme solution before the latter is mixed into the oil.

After completion of the enzyme treatment, the enzyme solution, togetherwith the decomposition products of the NHP contained in it, can beseparated from the oil phase, in batches or continuously, e.g., by meansof centrifugation. Since the enzymes are characterized by a high levelof stability and the amount of the decomposition products contained inthe solution is slight (they may precipitate as sludge) the same aqueousenzyme phase can be used several times. There is also the possibility offreeing the enzyme of the sludge, see, e.g., DE-A 43 39 556, so that anenzyme solution which is essentially free of sludge can be used again.In one aspect of this degumming process, oils which contain less than 15ppm phosphorus are obtained. One goal is phosphorus contents of lessthan 10 ppm; or, less than 5 ppm. With phosphorus contents below 10 ppm,further processing of the oil according to the process of distillativede-acidification is easily possible. A number of other ions, such asmagnesium, calcium, zinc, as well as iron, can be removed from the oil,e.g., below 0.1 ppm. Thus, this product possesses ideal prerequisitesfor good oxidation resistance during further processing and storage.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention also can also be used forreducing the amount of phosphorous-containing components in vegetableand animal oils as described, e.g., in EP patent EP 0513709. In thismethod, the content of phosphorus-containing components, especiallyphosphatides, such as lecithin, and the iron content in vegetable andanimal oils, which have previously been deslimed, e.g. soya oil, arereduced by enzymatic breakdown using a phospholipase A1, A2 or B of theinvention.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used for refining fator oils as described, e.g., in JP 06306386. The invention providesprocesses for refining a fat or oil comprising a step of converting aphospholipid in a fat or an oil into a water-solublephosphoric-group-containing substance and removing this substance. Theaction of an enzyme of the invention (e.g., a PLC) is utilized toconvert the phospholipid into the substance. Thus, it is possible torefine a fat or oil without carrying out an alkali refining step fromwhich industrial wastes containing alkaline waste water and a largeamount of oil are produced Improvement of yields can be accomplishedbecause the loss of neutral fat or oil from escape with the wastes canbe reduced to zero. In one aspect, gummy substances are converted intowater-soluble substances and removed as water-soluble substances byadding an enzyme of the invention having a phospholipase C activity inthe stage of degumming the crude oil and conducting enzymatic treatment.In one aspect, the phospholipase C of the invention has an activity thatcuts ester bonds of glycerin and phosphoric acid in phospholipids. Ifnecessary, the method can comprise washing the enzyme-treated oil withwater or an acidic aqueous solution. In one aspect, the enzyme of theinvention is added to and reacted with the crude oil. The amount ofphospholipase C employed can be 10 to 10,000 units, or, about 100 to2,000 units, per 1 kg of crude oil.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used forwater-degumming processes as described, e.g., in Dijkstra, Albert J., etal., Oleagineux, Corps Gras, Lipides (1998), 5(5), 367-370. In thisexemplary method, the water-degumming process is used for the productionof lecithin and for dry degumming processes using a degumming acid andbleaching earth. This method may be economically feasible only for oilswith a low phosphatide content, e.g., palm oil, lauric oils, etc. Forseed oils having a high NHP-content, the acid refining process is used,whereby this process is carried out at the oil mill to allow gumdisposal via the meal. In one aspect, this acid refined oil is apossible “polishing” operation to be carried out prior to physicalrefining.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used for degummingprocesses as described, e.g., in Dijkstra, et al., Res. Dev. Dep., N.V.Vandemoortele Coord. Cent., Izegem, Belg. JAOCS, J. Am. Oil Chem. Soc.(1989), 66:1002-1009. In this exemplary method, the total degummingprocess involves dispersing an acid such as H₃PO₄ or citric acid intosoybean oil, allowing a contact time, and then mixing a base such ascaustic soda or Na silicate into the acid-in-oil emulsion. This keepsthe degree of neutralization low enough to avoid forming soaps, becausethat would lead to increased oil loss. Subsequently, the oil passed to acentrifugal separator where most of the gums are removed from the oilstream to yield a gum phase with minimal oil content. The oil stream isthen passed to a second centrifugal separator to remove all remaininggums to yield a dilute gum phase, which is recycled. Washing and dryingor in-line alkali refining complete the process. After the adoption ofthe total degumming process, in comparison with the classical alkalirefining process, an overall yield improvement of about 0.5% isrealized. The totally degummed oil can be subsequently alkali refined,bleached and deodorized, or bleached and physically refined.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used for the removalof nonhydratable phospholipids from a plant oil, e.g., soybean oil, asdescribed, e.g., in Hvolby, et al., Sojakagefabr., Copenhagen, Den., J.Amer. Oil Chem. Soc. (1971) 48:503-509. In this exemplary method,water-degummed oil is mixed at different fixed pH values with buffersolutions with and without Ca⁺⁺, Mg/Ca-binding reagents, andsurfactants. The nonhydratable phospholipids can be removed in anonconverted state as a component of micelles or of mixed emulsifiers.Furthermore, the nonhydratable phospholipids are removable by conversioninto dissociated forms, e.g., by removal of Mg and Ca from thephosphatidates, which can be accomplished by acidulation or by treatmentwith Mg/Ca-complexing or Mg/Ca-precipitating reagents. Removal orchemical conversion of the nonhydratable phospholipids can result inreduced emulsion formation and in improved separation of the deacidifiedoil from the emulsion layer and the soapstock.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used for thedegumming of vegetable oils as described, e.g., Buchold, et al.,Frankfurt/Main, Germany. Fett Wissenschaft Technologie (1993), 95(8),300-304. In this exemplary process of the invention for the degumming ofedible vegetable oils, aqueous suspensions of an enzyme of theinvention, e.g., phospholipase A2, is used to hydrolyze the fatty acidbound at the sn2 position of the phospholipid, resulting in1-acyl-lysophospholipids which are insoluble in oil and thus moreamenable to physical separation. Even the addition of small amountscorresponding to about 700 lecitase units/kg oil results in a residual Pconcentration of less than 10 ppm, so that chemical refining isreplaceable by physical refining, eliminating the necessity forneutralization, soapstock splitting, and wastewater treatment.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used for thedegumming of vegetable oils as described, e.g., by EnzyMax, Dahlke,Klaus. Dept. G-PDO, Lurgi Ol-Gas, Chemie, GmbH, Frankfurt, Germany;Oleagineux, Corps Gras, Lipides (1997), 4(1), 55-57. This exemplaryprocess is a degumming process for the physical refining of almost anykind of oil. By an enzymatic-catalyzed hydrolysis, phosphatides areconverted to water-soluble lysophosphatides which are separated from theoil by centrifugation. The residual phosphorus content in theenzymatically degummed oil can be as low as 2 ppm P.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used for thedegumming of vegetable oils as described, e.g., by Cleenewerck, et al.,N. V. Vamo Mills, Izegem, Belg. Fett Wissenschaft Technologie (1992),94:317-22; and, Clausen, Kim; Nielsen, M., Novozymes A/S, Den. DanskKemi (2002) 83(2):24-27. The phospholipases and methods of the inventioncan incorporate the pre-refining of vegetable oils with acids asdescribed, e.g., by Nilsson-Johansson, et al., Fats Oils Div.,Alfa-Laval Food Eng. AB, Tumba, Swed. Fett Wissenschaft Technologie(1988), 90(11), 447-51; and, Munch, Ernst W. Cereol Deutschland GmbH,Mannheim, Germany. Editor(s): Wilson, Richard F., Proceedings of theWorld Conference on Oilseed Processing Utilization, Cancun, Mexico, Nov.12-17, 2000 (2001), Meeting Date 2000, 17-20.

The enzymes (e.g., lipases, phospholipases, esterases, proteases) of theinvention and methods of the invention can also be used for thedegumming of vegetable oils as described, e.g., by Jerzewska, et al.,Inst. Przemyslu Miesnego i Tluszczowego, Warsaw, Pol., Tluszcze Jadalne(2001), 36(3/4), 97-110. In this process of the invention, enzymaticdegumming of hydrated low-erucic acid rapeseed oil is by use of aphospholipase A2 of the invention. The enzyme can catalyze thehydrolysis of fatty acid ester linkages to the central carbon atom ofthe glycerol moiety in phospholipids. It can hydrolyze non-hydratablephospholipids to their corresponding hydratable lyso-compounds. With anonpurified enzyme preparation, better results can be achieved with theaddition of 2% preparation for 4 hours (87% P removal).

In another exemplary process of the invention for oil degumming (or anoil degumming process using an enzyme of the invention), an acidicpolymer, e.g., an alginate or pectin, is added. In this oil degummingprocess of the invention, an acidic polymer (e.g. alginic acid or pectinor a more soluble salt form) is added to the crude oil with a low amountof water (e.g., in a range of between about 0.5 to 5%). In this aspect,the acidic polymers can reduce and/or disrupt phospholipid-metalcomplexes by binding calcium and/or magnesium in the crude oil, therebyimproving the solubility of nonhydratable phospholipids. In one aspect,these phospholipids will enter the aqueous phase and either be convertedto diacylglycerol and the corresponding side chain or the intactphospholipid will be removed by subsequent centrifugation as a componentof the heavy phase. The presence of the acidic polymer in the aqueousphase can also increase the density of the aqueous phase and result inan improved separation of the heavy phase from the oil (light) phase.

One exemplary process of the invention for oil degumming (or an oildegumming process using an enzyme of the invention) alters thedeodorization procedure to get a diacylglycerol (DAG) fraction. Inalternative aspect, if necessary or desired, following enzyme-assisteddegumming, the deodorization conditions (temperature, pressure,configuration of the distillation apparatus) can be modified with thegoal of improving the separation of the free fatty acids (FFA) from thediacylglycerol/triacylglycerol fraction or further modified to separatethe diacylglycerol from the triacylglycerol fraction. As a result ofthese modifications, using this method of the invention, it is possibleto obtain food grade FFA and diacylglycerol if a hydrolase of theinvention (e.g., a phosphatase, or, a PLC or a combination of PLC andphosphatases) are used to degum edible oil in a physical refiningprocess.

In various aspects, practicing the methods of the invention as describedherein (or using the enzymes of the invention), have advantages such as:decrease or eliminate solvent and solvent recovery; lower capital costs;decrease downstream refining costs, decrease chemical usage, equipment,process time, energy (heat) and water usage/wastewater generation;produce higher quality oil; expeller pressed oil may be used withoutrefining in some cooking and sautéing applications (this pressed oil mayhave superior stability, color and odor characteristics and hightocopherol content); produce higher quality meal; produce a lower fatcontent in meal (currently, meal coming out of mechanical press causesdigestion problems in ruminants); produce improved nutritionalattributes—reduced levels of glucosinolates, tannins, sinapine, phyticacid (as described, e.g., in Technology and Solvents for ExtractingOilseeds and Nonpetroleum Oils, AOCS 1997).

In one aspect, the invention provides methods for refining vegetableoils (e.g., soybean oil, corn oil, cottonseed oil, palm oil, peanut oil,rapeseed oil, safflower oil, sunflower seed oil, sesame seed oil, ricebran oil, coconut oil or canola oil) and their byproducts, and processesfor deodorizing lecithin, for example, as described in U.S. Pat. No.6,172,248, or 6,172,247, wherein the methods comprise use of at leastone hydrolase of the invention, e.g., a phospholipase, such as aphospholipase C of the invention. Thus, the invention provides lecithinand vegetable oils comprising at least one enzyme of the invention. Inan exemplary organic acid refining process, vegetable oil is combinedwith a dilute aqueous organic acid solution and subjected to high shearto finely disperse the acid solution in the oil. The resultingacid-and-oil mixture is mixed at low shear for a time sufficient tosequester contaminants into a hydrated impurities phase, producing apurified vegetable oil phase. In this exemplary process, a mixer orrecycle system (e.g., recycle water tank) and/or a phosphatide orlecithin storage tank can be used, e.g., as described in U.S. Pat. Nos.4,240,972, 4,049,686, 6,172,247 or 6,172,248. These processes can beconducted as a batch or continuous process. Crude or degummed vegetableoil can be supplied from a storage tank (e.g., through a pump) and canbe heated. The vegetable oil to be purified can be either crude or“degummed” oil.

In one aspect, hydrolase enzymes such as the phosphatidylinositol-PLC(PI-PLC) enzymes of the invention are used for vegetable oil degummingHydrolase enzymes of the invention having PI-PLC activity can be usedalone or in combination with other enzymes (for instance PLC, PLD,phosphatase enzymes of the invention) to improve oil yield during thedegumming of vegetable oils (including soybean, canola, and sunflower).The PI-PLC enzymes of the invention may preferentially convertphosphatidylinositol to 1, 2-diacylglycerol (DAG) and phosphoinositolbut it may also demonstrate activity on other phospholipids includingphosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, orphosphatidic acid. The improvement in yield will be realized as anincrease in the amount of DAG in the enzyme-treated vegetable oil and anincrease in neutral oil, due to a decrease in the amount of oilentrained in the smaller gum fraction that results from enzyme treatmentof the vegetable oil.

Purification of Phytosterols from Vegetable Oils

The invention provides methods for purification of phytosterols andtriterpenes, or plant sterols, from vegetable oils using the enzymes ofthe invention.

Phytosterols that can be purified using enzymes (e.g., phospholipases)and methods of the invention include β-sitosterol, campesterol,stigmasterol, stigmastanol, β-sitostanol, sitostanol, desmosterol,chalinasterol, poriferasterol, clionasterol and brassicasterol. Plantsterols are important agricultural products for health and nutritionalindustries. Thus, enzymes and methods of the invention are used to makeemulsifiers for cosmetic manufacturers and steroidal intermediates andprecursors for the production of hormone pharmaceuticals. Enzymes andmethods of the invention are used to make (e.g., purify) analogs ofphytosterols and their esters for use as cholesterol-lowering agentswith cardiologic health benefits. Enzymes and methods of the inventionare used to purify plant sterols to reduce serum cholesterol levels byinhibiting cholesterol absorption in the intestinal lumen. Enzymes andmethods of the invention are used to purify plant sterols that haveimmunomodulating properties at extremely low concentrations, includingenhanced cellular response of T lymphocytes and cytotoxic ability ofnatural killer cells against a cancer cell line. Enzymes and methods ofthe invention are used to purify plant sterols for the treatment ofpulmonary tuberculosis, rheumatoid arthritis, management of HIV-infestedpatients and inhibition of immune stress, e.g., in marathon runners.

Enzymes and methods of the invention are used to purify sterolcomponents present in the sterol fractions of commodity vegetable oils(e.g., coconut, canola, cocoa butter, corn, cottonseed, linseed, olive,palm, peanut, rice bran, safflower, sesame, soybean, sunflower oils),such as sitosterol (40.2-92.3%), campesterol (2.6-38.6%), stigmasterol(0-31%) and 5-avenasterol (1.5-29%).

Methods of the invention can incorporate isolation of plant-derivedsterols in oil seeds by solvent extraction with chloroform-methanol,hexane, methylene chloride, or acetone, followed by saponification andchromatographic purification for obtaining enriched total sterols.Alternatively, the plant samples can be extracted by supercritical fluidextraction with supercritical carbon dioxide to obtain total lipidextracts from which sterols can be enriched and isolated. For subsequentcharacterization and quantification of sterol compounds, the crudeisolate can be purified and separated by a wide variety ofchromatographic techniques including column chromatography (CC), gaschromatography, thin-layer chromatography (TLC), normal phasehigh-performance liquid chromatography (HPLC), reversed-phase HPLC andcapillary electrochromatography. Of all chromatographic isolation andseparation techniques, CC and TLC procedures employ the most accessible,affordable and suitable for sample clean up, purification, qualitativeassays and preliminary estimates of the sterols in test samples.

Phytosterols are lost in the vegetable oils lost as byproducts duringedible oil refining processes. Phospholipases and methods of theinvention use phytosterols isolated from such byproducts to makephytosterol-enriched products isolated from such byproducts. Phytosterolisolation and purification methods of the invention can incorporate oilprocessing industry byproducts and can comprise operations such asmolecular distillation, liquid-liquid extraction and crystallization.

Methods of the invention can incorporate processes for the extraction oflipids to extract phytosterols. For example, methods of the inventioncan use nonpolar solvents as hexane (commonly used to extract most typesof vegetable oils) quantitatively to extract free phytosterols andphytosteryl fatty-acid esters. Steryl glycosides and fatty-acylatedsteryl glycosides are only partially extracted with hexane, andincreasing polarity of the solvent gave higher percentage of extraction.One procedure that can be used is the Bligh and Dyer chloroform-methanolmethod for extraction of all sterol lipid classes, includingphospholipids. One exemplary method to both qualitatively separate andquantitatively analyze phytosterol lipid classes comprises injection ofthe lipid extract into HPLC system.

Enzymes and methods of the invention can be used to remove sterols fromfats and oils, as described, e.g., in U.S. Pat. No. 6,303,803. This is amethod for reducing sterol content of sterol-containing fats and oils.It is an efficient and cost effective process based on the affinity ofcholesterol and other sterols for amphipathic molecules that formhydrophobic, fluid bilayers, such as phospholipid bilayers. Aggregatesof phospholipids are contacted with, for example, a sterol-containingfat or oil in an aqueous environment and then mixed. The molecularstructure of this aggregated phospholipid mixture has a high affinityfor cholesterol and other sterols, and can selectively remove suchmolecules from fats and oils. The aqueous separation mixture is mixedfor a time sufficient to selectively reduce the sterol content of thefat/oil product through partitioning of the sterol into the portion ofphospholipid aggregates. The sterol-reduced fat or oil is separated fromthe aqueous separation mixture. Alternatively, the correspondinglysterol-enriched fraction also may be isolated from the aqueousseparation mixture. These steps can be performed at ambienttemperatures, costs involved in heating are minimized, as is thepossibility of thermal degradation of the product. Additionally, aminimal amount of equipment is required, and since all requiredmaterials are food grade, the methods require no special precautionsregarding handling, waste disposal, or contamination of the finalproduct(s).

Enzymes and methods of the invention can be used to remove sterols fromfats and oils, as described, e.g., in U.S. Pat. No. 5,880,300.Phospholipid aggregates are contacted with, for example, asterol-containing fat or oil in an aqueous environment and then mixed.Following adequate mixing, the sterol-reduced fat or oil is separatedfrom the aqueous separation mixture. Alternatively, the correspondinglysterol-enriched phospholipid also may be isolated from the aqueousseparation mixture. Plant (e.g., vegetable) oils contain plant sterols(phytosterols) that also may be removed using the methods of the presentinvention. This method is applicable to a fat/oil product at any stageof a commercial processing cycle. For example, the process of theinvention may be applied to refined, bleached and deodorized oils (“RBDoils”), or to any stage of processing prior to attainment of RBD status.Although RBD oil may have an altered density compared to pre-RBD oil,the processes of the are readily adapted to either RBD or pre-RBD oils,or to various other fat/oil products, by variation of phospholipidcontent, phospholipid composition, phospholipid:water ratios,temperature, pressure, mixing conditions, and separation conditions asdescribed below.

Alternatively, the enzymes and methods of the invention can be used toisolate phytosterols or other sterols at intermediate steps in oilprocessing. For example, it is known that phytosterols are lost duringdeodorization of plant oils. A sterol-containing distillate fractionfrom, for example, an intermediate stage of processing can be subjectedto the sterol-extraction procedures described above. This provides asterol-enriched lecithin or other phospholipid material that can befurther processed in order to recover the extracted sterols.

Nutraceuticals

In one aspect, the compositions and methods of the invention can be usedto make nutraceuticals by processing or synthesizing lipids and oilsusing the enzymes of the invention, e.g., esterases, acylases, lipases,phospholipases or proteases of the invention. In one aspect, theprocessed or synthesized lipids or oils include poly-unsaturated fattyacids (PUFAs), diacylglycerides, e.g., 1,3-diacyl glycerides (DAGs),monoacylglycerides, e.g., 2-monoacylglycerides (MAGs) andtriacylglycerides (TAGs). In one aspect, the nutraceuticals is made byprocessing diacylglycerides, e.g., 1,3-diacyl glycerides (DAGs),monoacylglycerides, e.g., 2-monoacylglycerides (MAGs) and/ortriacylglycerides (TAGs) from plant (e.g., oilseed) sources or fromanimal (e.g., fish oil) sources.

In one aspect, the compositions and methods of the invention can be usedto fortify dietary compositions, especially cow's milk based products,e.g., cow's milk-based infant formulas, with bile salt-activatedhydrolases. The compositions made by the methods and compositions of theinvention can be used to feed newborn and premature infants, includingadministration of a bile salt-activated hydrolase of the invention toincrease fat digestion and therefore growth rate. Similarly, theinvention provides compositions and methods for treating subjects forinadequate pancreatic enzyme production by administration of bilesalt-activated hydrolase in conjunction with ingestion of fats; see alsodiscussion, below.

In one aspect, the invention provides a dietary composition comprising ahydrolase of the invention, e.g., bile salt-activated hydrolase of theinvention. In one aspect, the invention provides a dietary compositioncomprising a nutritional base comprising a fat and an effective amountof bile salt-activated hydrolase of the invention. In one aspect, theinvention provides a cow's milk-based infant formula comprising ahydrolase of the invention, e.g., bile salt-activated hydrolase of theinvention. In one aspect, the hydrolase of the invention is active inthe digestion of long chain fatty acids, e.g., C₁₂ to C₂₂, which make upa very high percentage of most milks, e.g., 99% of human breast milk.See, e.g., U.S. Pat. No. 5,000,975.

In one aspect, the invention provides a dietary composition comprising avegetable oil fat and a hydrolase of the invention. The inventionprovides methods of processing milk based products and/or vegetableoil-comprising compositions to make dietary compositions. In one aspect,the processed compositions comprise a lauric acid oil, an oleic acidoil, a palmitic acid oil and/or a linoleic acid oil. In one aspect, arice bran oil, sunflower oleic oil and/or canola oil may be used asoleic acids oils. In one aspect, fats and oils, e.g., oilseeds, fromplants, including, e.g., rice, canola, sunflower, olive, palm, soy orlauric type oils for use in the nutraceuticals and dietary compositionsare processed or made using a hydrolase of the invention. See, e.g.,U.S. Pat. No. 4,944,944.

In one aspect, the enzymes of the invention are provided in a form thatis stable to storage in the formula and/or the stomach, but active whenthe formulation reaches the portion of the gastrointestinal tract wherethe formula would normally be digested. Formulations (e.g.,microcapsules) for release in the intestine are well known in the art,e.g., biodegradable polymers such as polylactide and polyglycolide, asdescribed, e.g., in U.S. Pat. Nos. 4,767,628; 4,897,268; 4,925,673;5,902,617.

Confectionaries, Cacao Butter and Foods

In one aspect, the compositions and methods of the invention can be usedto make and process hard butters, such as cacao butter (cocao butter).The compositions and methods of the invention can be used to make cocoabutter alternatives by “structured” synthetic techniques using theenzymes of the invention, e.g., esterases, acylases, lipases,phospholipases or proteases of the invention. For example, in oneaspect, the methods of the invention process or synthesizetriacylglycerides, diacylglycerides and/or monoacylglycerides for useas, e.g., cocoa butter alternatives. In one aspect, the methods of theinvention generate a hard butter with a defined “plastic region” tomaintain sufficient hardness below or at room temperature. In oneaspect, the processed or synthesized lipid is designed to have a verynarrow “plastic region,” e.g., in one aspect, where it rapidly melts atabout body temperature. Natural cacao butter begins to soften atapproximately 30° C. to 32° C., and completely melts at approximately36° C. Natural cacao butter can contain 70 wt % or more of three1,3-disaturated-2-oleoyl glycerols, which are 1,3-dipalmitoyl-2-oleoylglycerol (POP), 1-palmitoyl-2-oleoyl glycerol (POSt) and1,3-distearoyl-2-oleoyl glycerol (StOSt). These three glycerols show asimilar melting behavior to each other and are responsible for meltingproperties of the cacao butter, exhibiting a very narrow plastic region.The invention provides synthetic cacao butters or processed cacaobutters (synthesized or processed using a hydrolase of the invention,all possible composition are referred to as cocoa-butter alternatives)with varying percentages of, 3-dipalmitoyl-2-oleoyl glycerol (POP),1-palmitoyl-2-oleoyl glycerol (POSt) and 1,3-distearoyl-2-oleoylglycerol (StOSt), depending on the desired properties of the syntheticcacao butter, and, synthetic cacao butters with more or less than 70 wt% of the three 1,3-disaturated-2-oleoyl glycerols. The synthetic cacaobutters of the invention can partially or completely replace natural orunprocessed cacao butters and can maintain or improve essential hardbutter properties.

The invention provides synthetic cacao butters or processed cacaobutters (synthesized or processed using a hydrolase of the invention)with desired properties for use in confectionary, bakery andpharmaceutical products. In one aspect, the invention providesconfectionary, bakery and pharmaceutical products comprising a hydrolaseof the invention. In one aspect, the methods of the invention make orprocess a lipid (a fat) from a confection (e.g., a chocolate) or to beused in a confection. In one aspect, a lipid is made or processed suchthat the chocolate shows less finger-imprinting than chocolate made fromnatural cocoa butter, while still having sharp melting characteristicsin the mouth. In one aspect, a lipid is made or processed such that aconfection (e.g., chocolate) can be made at a comparatively high ambienttemperature, or, be made using a cooling water at a comparatively hightemperature. In one aspect, the lipid is made or processed such that aconfection (e.g., chocolate) can be stored under relatively warmerconditions, e.g., tropical or semi-tropical conditions or in centrallyheated buildings. In one aspect, the lipids are made or processed suchthat a confection (e.g., chocolate) will have a lipid (fat) content ofconsistent composition and quality. The enzymes of the invention can beused to provide a substitute composition for cacao butter which cansignificantly improve its thermal stability and replace it in a widerange of applications.

Margarine and Shortening Production

The invention provides synthetic or processed fats, e.g., margarine andshortening synthesized or processed using a hydrolase of the invention.In one aspect, the invention provides processed fats comprising avegetable oil, such as soybean oil, corn oil, rapeseed oil, palm oil orlauric type oils synthesized or processed using a hydrolase of theinvention. The synthetic or processed fats, e.g., margarine andshortening, are designed to have a desired “plasticity.” Many of theplastic fat products, such as margarine and shortening, are producedfrom hard stocks and liquid oils as raw materials. For example, liquidoils such as soybean oil, corn oil, palm oil and rapeseed oil, areblended with their hardened oils (hard stocks), and the blend isadjusted to have an appropriate consistency (plasticity). The plasticfat products such as margarine and shortening so produced tend to causethe formation of relatively coarse crystallines because fats and oilsused as the raw materials are composed of fatty acids having almost thesame carbon chain length. In other words, they have a highly-unifiedcomposition of fatty acids. For this reason, the plasticity of theseproducts can be maintained at an appropriate degree only within a narrowtemperature range, so that the liquid oils contained therein have atendency to exude. In one aspect, the invention provides methods ofmaking or processing fats designed such that they have a varied (anddefined) composition of fatty acids. The resultant oil, e.g., margarineor shortening, can have a broader range of plasticity.

In one aspect, the methods and compositions of the invention are used tomake or process vegetable oils, such as soybean oil, corn oil, rapeseedoil, palm oil or lauric type oils using the hydrolases of the invention,including inter-esterification and enzymatic transesterification, seee.g., U.S. Pat. No. 5,288,619. The methods and compositions of theinvention can be used in place of random inter-esterification asdescribed in, e.g., U.S. Pat. No. 3,949,105. In one aspect, the methodsand compositions of the invention are used to in enzymatictransesterification for preparing an oil, e.g., a margarine oil, havingboth low trans-acid and low intermediate chain fatty acid content.

In one aspect, the symmetric structure of an oil, e.g., a palm or laurictype oils is modified, e.g., into a random structure. Thus, the methodsof the invention can be used to modify the properties of plastic fatproducts. In one aspect, the modification of oils by the methods of theinvention can be designed to prevent or slow gradually hardening of theoil with time, particularly when the products are being stored.

In one aspect, the methods and compositions of the invention in atrans-esterification reaction mixture comprising a stearic acid sourcematerial and an edible liquid vegetable oil, trans-esterifying thestearic acid source material and the vegetable oil using a 1-,3-positionally specific lipase of the invention, and then hydrogenatingthe fatty acid mixture to provide a recycle stearic acid source materialfor a recyclic reaction with the vegetable oil. See e.g., U.S. Pat. No.5,288,619.

In one aspect, an inter-esterification reaction is conducted with alipase of the invention. In one aspect, the lipase of the invention hasa selectivity for the 1- and 3-positions of triglyceride to slow orinhibit an increase in the amount of tri-saturated triglycerides in theoil. In this reaction of the invention, deficiencies of conventionalrandom inter-esterification and the difficulty of inter-esterificationwith a non-specific lipase can be overcome because theinter-esterification is conducted by an enzyme of the invention having aspecificity for the 1- and 3-positions of triglycerides. In one aspect,the exudation of liquid oils contained in the products is slowed orprevented with a temperature increase in the reaction to inhibit a risein the melting point caused by an increase in the amount oftri-saturated triglycerides. This addresses the problem of hardening ofproducts during long-term storage.

Latex Processing

The methods and compositions (e.g., enzymes of the invention, e.g.,esterases, acylases, lipases, phospholipases or proteases of theinvention) of the invention can be used to selectively hydrolyzesaturated esters over unsaturated esters into acids or alcohols. In oneaspect, the invention provides for the selective hydrolysis of ethylpropionate over ethyl acrylate. In one aspect, these methods are used toremove undesired esters from monomer feeds used in latex polymerizationand from the latexes after polymerization. The methods and compositions(hydrolases) of the invention can be used to treat latexes for a varietyof purposes, e.g., to treat latexes used in hair fixative compositionsto remove unpleasant odors. Latexes treated by the methods andcompositions of the invention include, e.g., polymers containingacrylic, vinyl and unsaturated acid monomers, including alkyl acrylatemonomers such as methyl acrylate, ethyl acrylate, propyl acrylate andbutyl acrylate, and acrylate acids such as acrylic acid, methacrylicacid, crotonic acid, itaconic acid and mixtures thereof. See, e.g., U.S.Pat. No. 5,856,150.

Treating Hydrolase Deficiencies

The methods and compositions (enzymes of the invention, e.g., esterases,acylases, lipases, phospholipases or proteases of the invention) of theinvention can be used in the treatment of a hydrolase deficiency in ananimal, e.g., a mammal, such as a human. For example, in one aspect, themethods and compositions of the invention are used to treat patientssuffering from a deficiency of a pancreatic lipase. In one aspect, thelipase is administered orally. An enzyme of the invention can bedelivered in place of or with a preparation of pig pancreas enzyme.

In one aspect, the compositions of the invention used for thesetreatments are active under acidic conditions. In one aspect, thecompositions of the invention are administered orally in formulations(e.g., tablets) that pass through the acid regions of the stomach anddischarge the enzyme only in the relatively alkaline environment of thejejunum. In one aspect, a hydrolase of the invention is formulated witha carrier such as lactose, saccharose, sorbitol, mannitol, starch,cellulose derivatives or gelatine or any other such excipient. Alubricant such as magnesium stearate, calcium stearate or polyethyleneglycol wax also can be added. A concentrated sugar solution, which maycontain additives such as talc, titanium dioxide, gelatine or gumArabic, can be added as a coating. Soft or hard capsules can be used toencapsulate a hydrolase as a liquid or as a solid preparation. See,e.g., U.S. Pat. Nos. 5,691,181; 5,858,755.

Detergents

The methods and compositions (enzymes of the invention, e.g., esterases,acylases, lipases, phospholipases or proteases of the invention) of theinvention can be used in making and using detergents. A hydrolase of theinvention can be added to, e.g., be blended with, any known detergentcomposition, solid or liquid, with or without changing the compositionof the detergent composition. For examples, a hydrolase of the inventioncan be added to any soap, e.g., aliphatic sulfates such as straight orbranched chain alkyl or alkenyl sulfates, amide sulfates, alkyl oralkenyl ether sulfates having a straight or branched chain alkyl oralkenyl group to which one or more of ethylene oxide, propylene oxideand butylene oxide added, aliphatic sulfonates such as alkyl sulfonates,amide sulfonates, dialkyl sulfosuccinates, sulfonates of alpha-olefins,of vinylidene-type olefins and of internal olefins, aromatic sulfonatessuch as straight or branched chain alkylbenzenesulfonates, alkyl oralkenyl ether carbonates or amides having a straight or branched chainalkyl or alkenyl group to which one or more of ethylene oxide, propyleneoxide and butylene oxide added, or amides, alpha-sulfo-fatty acid saltsor esters, amino acid type surfactants, phosphate surfactants such asalkyl or alkenyl acidic phosphates, and alkyl or alkenyl phosphates,sulfonic acid type amphoteric surfactants, betaine type amphotericsurfactants, alkyl or alkenyl ethers or alcohols having a straight orbranched chain alkyl or alkenyl group to which one or more of ethyleneoxide, propylene oxide and butylene oxide added, polyoxy-ethylenealkylphenyl ethers having a straight or branched chain alkyl group to whichone or more of ethylene oxide, propylene oxide and butylene oxide added,higher fatty acid alkanolamides or alkylene oxide adducts thereof,sucrose fatty acid esters, fatty acid glycerol monoesters, alkyl- oralkenyl-amine oxides, tetraalkyl-ammonium salt type cationicsurfactants, or a combination thereof. See, e.g., U.S. Pat. No.5,827,718.

The invention provides detergent compositions comprising one or morepolypeptides (hydrolases) of the invention. Surface-active and/ornon-surface-active forms can be used. In one aspect, the amount of totalhydrolase, surface-active and/or non-surface-active, used in theinvention can be from about 0.0001% to about 1.0%, or from about 0.0002%to about 0.5%, by weight, of the detergent composition. In one aspect,of the detergent composition, the surface-active hydrolase is from about5% to about 67% and the non-surface-active hydrolase is from about 33%to about 95% of the total hydrolase activity in the enzymatic mixture.In one aspect, the optimum pH of the total enzymatic mixture is betweenabout 5 to about 10.5.

In one aspect, the detergent compositions of the invention includealkaline hydrolases of the invention which function at alkaline pHvalues, since the pH of a washing solution can be in an alkaline pHrange under ordinary washing conditions. See, e.g., U.S. Pat. No.5,454,971

The polypeptides of the invention (enzymes of the invention, e.g.,esterases, acylases, lipases, phospholipases or proteases of theinvention) can be used in any detergent composition, which are wellknown in the art, see, e.g., U.S. Pat. Nos. 5,069,810; 6,322,595;6,313,081. For example, in one aspect, a laundry detergent compositionis provided. It can comprise 0.8 ppm to 80 ppm of a lipase of theinvention.

The invention incorporates all methods of making and using detergentcompositions, see, e.g., U.S. Pat. Nos. 6,413,928; 6,399,561; 6,365,561;6,380,147.

The invention incorporates all methods of making and using detergentcompositions, see, e.g., U.S. Pat. Nos. 6,413,928; 6,399,561; 6,365,561;6,380,147. The detergent compositions can be a one and two part aqueouscomposition, a non-aqueous liquid composition, a cast solid, a granularform, a particulate form, a compressed tablet, a gel and/or a paste anda slurry form. The hydrolases of the invention can also be used as adetergent additive product in a solid or a liquid form. Such additiveproducts are intended to supplement or boost the performance ofconventional detergent compositions and can be added at any stage of thecleaning process.

The invention also provides methods capable of removing gross foodsoils, films of food residue and other minor food compositions usingthese detergent compositions. Hydrolases of the invention can facilitatethe removal of stains by means of catalytic hydrolysis of proteins.Hydrolases of the invention can be used in dishwashing detergents intextile laundering detergents.

The actual active enzyme content depends upon the method of manufactureof a detergent composition and is not critical, assuming the detergentsolution has the desired enzymatic activity. In one aspect, the amountof hydrolases present in the final solution ranges from about 0.001 mgto 0.5 mg per gram of the detergent composition. The particular enzymechosen for use in the process and products of this invention dependsupon the conditions of final utility, including the physical productform, use pH, use temperature, and soil types to be degraded or altered.The enzyme can be chosen to provide optimum activity and stability forany given set of utility conditions. In one aspect, the hydrolases ofthe present invention are active in the pH ranges of from about 4 toabout 12 and in the temperature range of from about 20° C. to about 95°C. The detergents of the invention can comprise cationic, semi-polarnonionic or zwitterionic surfactants; or, mixtures thereof.

Enzymes of the invention can be formulated into powdered and liquiddetergents having pH between 4.0 and 12.0 at levels of about 0.01 toabout 5% (preferably 0.1% to 0.5%) by weight. These detergentcompositions can also include other enzymes such as proteases,cellulases, lipases or endoglycosidases, endo-beta.-1,4-glucanases,beta-glucanases, endo-beta-1,3(4)-glucanases, cutinases, peroxidases,laccases, amylases, glucoamylases, pectinases, reductases, oxidases,phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,mannanases, xyloglucanases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, polygalacturonases,rhamnogalacturonases, galactanases, pectin lyases, pectinmethylesterases, cellobiohydrolases and/or transglutaminases. Thesedetergent compositions can also include builders and stabilizers.

The addition of hydrolases of the invention to conventional cleaningcompositions does not create any special use limitation. In other words,any temperature and pH suitable for the detergent is also suitable forthe compositions of the invention as long as the enzyme is active at ortolerant of the pH and/or temperature of the intended use. In addition,the proteases of the invention can be used in a cleaning compositionwithout detergents, again either alone or in combination with buildersand stabilizers.

The present invention provides cleaning compositions including detergentcompositions for cleaning hard surfaces, detergent compositions forcleaning fabrics, dishwashing compositions, oral cleaning compositions,denture cleaning compositions, and contact lens cleaning solutions.

In one aspect, the invention provides a method for washing an objectcomprising contacting the object with a polypeptide of the inventionunder conditions sufficient for washing. A hydrolase of the inventionmay be included as a detergent additive. The detergent composition ofthe invention may, for example, be formulated as a hand or machinelaundry detergent composition comprising a polypeptide of the invention.A laundry additive suitable for pre-treatment of stained fabrics cancomprise a polypeptide of the invention. A fabric softener compositioncan comprise a hydrolase of the invention. Alternatively, a hydrolasesof the invention can be formulated as a detergent composition for use ingeneral household hard surface cleaning operations. In alternativeaspects, detergent additives and detergent compositions of the inventionmay comprise one or more other enzymes such as a protease, a lipase, acutinase, another protease, a carbohydrase, a cellulase, a pectinase, amannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., alactase, and/or a peroxidase (see also, above). The properties of theenzyme(s) of the invention are chosen to be compatible with the selecteddetergent (i.e. pH-optimum, compatibility with other enzymatic andnon-enzymatic ingredients, etc.) and the enzyme(s) is present ineffective amounts. In one aspect, enzymes of the invention are used toremove malodorous materials from fabrics. Various detergent compositionsand methods for making them that can be used in practicing the inventionare described in, e.g., U.S. Pat. Nos. 6,333,301; 6,329,333; 6,326,341;6,297,038; 6,309,871; 6,204,232; 6,197,070; 5,856,164.

When formulated as compositions suitable for use in a laundry machinewashing method, the hydrolases of the invention can comprise both asurfactant and a builder compound. They can additionally comprise one ormore detergent components, e.g., organic polymeric compounds, bleachingagents, additional enzymes, suds suppressors, dispersants, lime-soapdispersants, soil suspension and anti-redeposition agents and corrosioninhibitors. Laundry compositions of the invention can also containsoftening agents, as additional detergent components. Compositionscontaining hydrolases of the invention can provide fabric cleaning,stain removal, whiteness maintenance, softening, color appearance, dyetransfer inhibition and sanitization when formulated as laundrydetergent compositions.

The density of the laundry detergent compositions of the invention canrange from about 200 to 1500 g/liter, or, about 400 to 1200 g/liter, or,about 500 to 950 g/liter, or, 600 to 800 g/liter, of composition; thiscan be measured at about 20° C.

The “compact” form of laundry detergent compositions of the invention isbest reflected by density and, in terms of composition, by the amount ofinorganic filler salt. Inorganic filler salts are conventionalingredients of detergent compositions in powder form. In conventionaldetergent compositions, the filler salts are present in substantialamounts, typically 17% to 35% by weight of the total composition. In oneaspect of the compact compositions, the filler salt is present inamounts not exceeding 15% of the total composition, or, not exceeding10%, or, not exceeding 5% by weight of the composition. The inorganicfiller salts can be selected from the alkali and alkaline-earth-metalsalts of sulphates and chlorides, e.g., sodium sulphate.

Liquid detergent compositions of the invention can also be in a“concentrated form.” In one aspect, the liquid detergent compositionscan contain a lower amount of water, compared to conventional liquiddetergents. In alternative aspects, the water content of theconcentrated liquid detergent is less than 40%, or, less than 30%, or,less than 20% by weight of the detergent composition. Detergentcompounds of the invention can comprise formulations as described in WO97/01629.

Hydrolases of the invention can be useful in formulating variouscleaning compositions. A number of known compounds are suitablesurfactants including nonionic, anionic, cationic, or zwitterionicdetergents, can be used, e.g., as disclosed in U.S. Pat. Nos. 4,404,128;4,261,868; 5,204,015. In addition, enzymes of the invention can be used,for example, in bar or liquid soap applications, dish care formulations,contact lens cleaning solutions or products, peptide hydrolysis, wastetreatment, textile applications, as fusion-cleavage enzymes in proteinproduction, and the like. Hydrolases of the invention may provideenhanced performance in a detergent composition as compared to anotherdetergent protease, that is, the enzyme group may increase cleaning ofcertain enzyme sensitive stains such as grass or blood, as determined byusual evaluation after a standard wash cycle. Hydrolases of theinvention can be formulated into known powdered and liquid detergentshaving pH between 6.5 and 12.0 at levels of about 0.01 to about 5% (forexample, about 0.1% to 0.5%) by weight. These detergent cleaningcompositions can also include other enzymes such as other knownesterases, phospholipases, proteases, amylases, cellulases, lipases orendoglycosidases, as well as builders and stabilizers.

Processes for Coating and Finishing Fabrics

The methods and compositions (enzymes of the invention, e.g., esterases,acylases, lipases, phospholipases or proteases of the invention) of theinvention can be used in processes for coating and finishing fabrics,fibers or yarns. In one aspect, insoluble cellulosic polymers arereacted with carboxylic acids or esters thereof in the presence of ahydrolase of the invention. The cellulosic polymer may be cotton,viscose, rayon, lyocell, flax, linen, ramie, and all blends thereof; andblends thereof with polyesters, wool, polyamides, acrylics andpolyacrylics. In alternative aspects, the methods and compositions(hydrolases) of the invention can be used in a softening finish process,i.e. improvement of the hand and drape of the final fabric, for dyeing apolymeric material, for obtaining flame retardancy, for obtaining waterrepellency, for obtaining brightness, e.g. optical brightness, of apolymeric material, and for obtaining resin finishing (“permanentpress”). In one aspect, the methods and compositions (hydrolases) of theinvention can be used for obtaining flame retardancy in a fabric using,e.g., a halogen-substituted carboxylic acid or an ester thereof, i.e. afluorinated, chlorinated or bromated carboxylic acid or an esterthereof. In one aspect, the processes are carried out under conditions(e.g. temperature, pH, solvent) that favors the esterification processover hydrolytic cleavage of an ester bend. In one aspect, theesterification process is carried out using water as a solvent, or, theprocess may be carried out without a solvent, or, the reaction may takeplace in a microemulsion formed by adding an carboxylic acid or an esterthereof to a mixture of water and a suitable surfactant. See, e.g., U.S.Pat. No. 5,733,750.

In one aspect, a hydrolase of the invention is absorbed or adsorbed orotherwise immobilized on a surface, such as a fabric, fiber or yarn. Inone aspect, a fabric-hydrolase complex is formed for, e.g., lipidremoval, e.g., food or oil stain removal. The hydrolase may be sorbed onthe fabric, fiber or yarn before or after staining. The active hydrolasecan hydrolyze the stain on dry fabric, fiber or yarn, or fabric, fiberor yarn in laundering solutions. In one aspect, a hydrolase of theinvention has enhanced stability to denaturation by surfactants and toheat deactivation. In the way, the hydrolase can be resistant to removalfrom the fabric, fiber or yarn during laundering, can retain substantialactivity after drying at an elevated temperature, and can retainactivity during fabric storage or wear. Redeposition of food, oil andoil hydrolysis by-products during laundering of fabric also can beretarded by a hydrolase of the invention. Oil hydrolysis by-products canbe removed during laundering of the fabric, e.g., at a basic pH or inthe presence of a surfactant. In one aspect, a hydrolase of theinvention is absorbed or adsorbed or otherwise immobilized on a gel,glass, plastic or metal solid as well as a fabric, fiber or yarn.

In alternative aspects, hydrolases of the invention are useful to treata wide variety of natural, synthetic or metallic fabrics, includingtextiles or woven or non-woven cloths, including nylon, polycotton,polyester, woven polyester, double knit polyester, silk, vinyl, cottonflannel, rayon velvet, acrylic felt, wool blend (polyester/wool),synthetic blend (polyester/polyurethane), latexes. Other surfaces thatcan be treated with a hydrolase of the invention include kitchen orcooking devices and utensils, e.g., pot cleaner materials such ascellulose sponge, nylon and stainless steel scrubbers and copper cloth,dishwashers, food storage devices, e.g., refrigerators, freezers and thelike.

The surfaces that have been treated in accordance with the invention canalready be stained by (or carrying) oil before an enzyme-fabric complexis formed or the complex can be formed before such exposure. Examples ofembodiments useful for the former applications include pre-wash liquidor gelled compositions that can be sprayed or directly applied tospecific areas of oily stains. The garments or linens can then be storedin a laundry hamper, for example, and laundered in the normal course ofa household's routine because degradation of the oily stain intohydrolysis by-products will be occurring during storage. Alternatively,fabric may be pretreated before use to convey improved oil stain removalproperties. In one aspect, a hydrolase is immobilized on surfaces tofacilitate oil removal from the surfaces and to alter wettability of thesurfaces. In one aspect, a hydrolase is adsorbed on a fabric before orafter an oil stain, and the hydrolase is active to hydrolyze an oilstain on dry fabric or fabric in laundering solutions. In one aspect,the sorbed hydrolase has enhanced stability to denaturation bysurfactants and to heat deactivation, is resistant to removal fromfabric during laundering, retains substantial activity after dryingfabric at an elevated temperature, and/or retains activity during fabricstorage or wear. In one aspect, redeposition of oil and oil hydrolysisby-products during laundering of fabric is retarded by the hydrolase. Inone aspect, oil hydrolysis by-products are removable during launderingof fabric at a basic pH or in the presence of a surfactant. See, e.g.,U.S. Pat. No. 6,265,191.

Treating Fibers and Textiles

The invention provides methods of treating fibers and fabrics using oneor more hydrolases of the invention. The hydrolases can be used in anyfiber- or fabric-treating method, which are well known in the art, see,e.g., U.S. Pat. Nos. 6,261,828; 6,077,316; 6,024,766; 6,021,536;6,017,751; 5,980,581; US Patent Publication No. 20020142438 A1. Forexample, hydrolases of the invention can be used in fiber and/or fabricdesizing. In one aspect, the feel and appearance of a fabric is improvedby a method comprising contacting the fabric with a hydrolase of theinvention in a solution. In one aspect, the fabric is treated with thesolution under pressure. For example, hydrolases of the invention can beused in the removal of stains.

In one aspect, hydrolases of the invention are applied during or afterthe weaving of textiles, or during the desizing stage, or one or moreadditional fabric processing steps. During the weaving of textiles, thethreads are exposed to considerable mechanical strain. Prior to weavingon mechanical looms, warp yarns are often coated with sizing starch orstarch derivatives in order to increase their tensile strength and toprevent breaking. The hydrolases of the invention can be applied toremove these sizing starch or starch derivatives. After the textileshave been woven, a fabric can proceed to a desizing stage. This can befollowed by one or more additional fabric processing steps. Desizing isthe act of removing “size” from textiles. After weaving, the sizecoating must be removed before further processing the fabric in order toensure a homogeneous and wash-proof result. The invention provides amethod of desizing comprising enzymatic treatment of the “size” by theaction of hydrolases of the invention.

The enzymes of the invention can be used to desize fabrics, includingcotton-containing fabrics, as detergent additives, e.g., in aqueouscompositions. The invention provides methods for producing a stonewashedlook on indigo-dyed denim fabric and garments. For the manufacture ofclothes, the fabric can be cut and sewn into clothes or garments. Thesecan be finished before or after the treatment. In particular, for themanufacture of denim jeans, different enzymatic finishing methods havebeen developed. The finishing of denim garment normally is initiatedwith an enzymatic desizing step, during which garments are subjected tothe action of amylolytic enzymes in order to provide softness to thefabric and make the cotton more accessible to the subsequent enzymaticfinishing steps. The invention provides methods of finishing denimgarments (e.g., a “bio-stoning process”), enzymatic desizing andproviding softness to fabrics using the hydrolases of the invention. Theinvention provides methods for quickly softening denim garments in adesizing and/or finishing process.

Other enzymes can be also be used in these desizing processes. Forexample, an alkaline and thermostable amylase and hydrolase can becombined in a single bath for desizing and bioscouring. Among advantagesof combining desizing and scouring in one step are cost reduction andlower environmental impact due to savings in energy and water usage andlower waste production. Exemplary application conditions for desizingand bioscouring are about pH 8.5 to 10.0 and temperatures of about 40°C. and up. Using a hydrolase of the invention, low enzyme dosages, e.g.,about 100 grams (g) per a ton of cotton, and short reaction times, e.g.,about 15 minutes, can be used to obtain efficient desizing and scouringwith out added calcium.

In one aspect, an alkaline and thermostable amylase and hydrolase arecombined in a single bath desizing and bioscouring. Among advantages ofcombining desizing and scouring in one step are cost reduction and lowerenvironmental impact due to savings in energy and water usage and lowerwaste production. Application conditions for desizing and bioscouringcan be between about pH 8.5 to pH 10.0 and temperatures at about 40° C.and up. Low enzyme dosages (e.g., about 100 g per a ton of cotton) andshort reaction times (e.g., about 15 minutes) can be used to obtainefficient desizing and scouring with out added calcium.

The hydrolases of the invention can be used in combination with othercarbohydrate degrading enzymes, e.g., cellulase, arabinanase,xyloglucanase, pectinase, and the like, for the preparation of fibers orfor cleaning of fibers. These can be used in combination withdetergents. In one aspect, hydrolases of the invention can be used intreatments to prevent the graying of a textile.

The hydrolases of the invention can be used to treat any cellulosicmaterial, including fibers (e.g., fibers from cotton, hemp, flax orlinen), sewn and unsewn fabrics, e.g., knits, wovens, denims, yarns, andtoweling, made from cotton, cotton blends or natural or manmadecellulosics (e.g. originating from xylan-containing cellulose fiberssuch as from wood pulp) or blends thereof. Examples of blends are blendsof cotton or rayon/viscose with one or more companion material such aswool, synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyesterfibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers,aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose,ramie, hemp, flax/linen, jute, cellulose acetate fibers, lyocell).

The textile treating processes of the invention (using hydrolases of theinvention) can be used in conjunction with other textile treatments,e.g., scouring and bleaching. Scouring is the removal of non-cellulosicmaterial from the cotton fiber, e.g., the cuticle (mainly consisting ofwaxes) and primary cell wall (mainly consisting of pectin, protein andxyloglucan). A proper wax removal is necessary for obtaining a highwettability. This is needed for dyeing. Removal of the primary cellwalls by the processes of the invention improves wax removal and ensuresa more even dyeing. Treating textiles with the processes of theinvention can improve whiteness in the bleaching process. The mainchemical used in scouring is sodium, hydroxide in high concentrationsand at high temperatures. Bleaching comprises oxidizing the textile.Bleaching typically involves use of hydrogen peroxide as the oxidizingagent in order to obtain either a fully bleached (white) fabric or toensure a clean shade of the dye.

The invention also provides alkaline hydrolases (hydrolases active underalkaline conditions). These have wide-ranging applications in textileprocessing, degumming of plant fibers (e.g., plant bast fibers),treatment of pectic wastewaters, paper-making, and coffee and teafermentations. See, e.g., Hoondal (2002) Applied Microbiology andBiotechnology 59:409-418.

Treating Foods and Food Processing

The hydrolases of the invention have numerous applications in foodprocessing industry. For example, in one aspect, the hydrolases of theinvention are used to improve the extraction of oil from oil-rich plantmaterial, e.g., oil-rich seeds, for example, soybean oil from soybeans,olive oil from olives, rapeseed oil from rapeseed and/or sunflower oilfrom sunflower seeds or rice bran oil.

The hydrolases of the invention can be used for separation of componentsof plant cell materials. For example, hydrolases of the invention can beused in the separation of protein-rich material (e.g., plant cells) intocomponents, e.g., sucrose from sugar beet or starch or sugars frompotato, pulp or hull fractions. In one aspect, hydrolases of theinvention can be used to separate protein-rich or oil-rich crops intovaluable protein and oil and hull fractions. The separation process maybe performed by use of methods known in the art.

The hydrolases of the invention can be used in the preparation of fruitor vegetable juices, syrups, extracts and the like to increase yield.The hydrolases of the invention can be used in the enzymatic treatment(e.g., hydrolysis of proteins) of various plant cell wall-derivedmaterials or waste materials, e.g. from wine or juice production, oragricultural residues such as vegetable hulls, bean hulls, sugar beetpulp, olive pulp, potato pulp, and the like. The hydrolases of theinvention can be used to modify the consistency and appearance ofprocessed fruit or vegetables. The hydrolases of the invention can beused to treat plant material to facilitate processing of plant material,including foods, facilitate purification or extraction of plantcomponents. The hydrolases of the invention can be used to improve feedvalue, decrease the water binding capacity, improve the degradability inwaste water plants and/or improve the conversion of plant material toensilage, and the like.

Animal Feeds and Food or Feed Additives

The invention provides methods for treating animal feeds and foods andfood or feed additives using hydrolases of the invention, animalsincluding mammals (e.g., humans), birds, fish and the like. Theinvention provides animal feeds, foods, and additives comprisinghydrolases of the invention. In one aspect, treating animal feeds, foodsand additives using hydrolases of the invention can help in theavailability of nutrients, e.g., starch, in the animal feed or additive.By breaking down difficult to digest proteins or indirectly or directlyunmasking starch (or other nutrients), the hydrolase makes nutrientsmore accessible to other endogenous or exogenous enzymes. The hydrolasecan also simply cause the release of readily digestible and easilyabsorbed nutrients and sugars.

Hydrolases of the present invention, in the modification of animal feedor a food, can process the food or feed either in vitro (by modifyingcomponents of the feed or food) or in vivo. Hydrolases can be added toanimal feed or food compositions containing high amounts ofarabinogalactans or galactans, e.g. feed or food containing plantmaterial from soy bean, rape seed, lupin and the like. When added to thefeed or food the hydrolase significantly improves the in vivo break-downof plant cell wall material, whereby a better utilization of the plantnutrients by the animal (e.g., human) is achieved. In one aspect, thegrowth rate and/or feed conversion ratio (i.e. the weight of ingestedfeed relative to weight gain) of the animal is improved. For example apartially or indigestible galactan-comprising protein is fully orpartially degraded by a hydrolase of the invention, e.g. in combinationwith another enzyme, e.g., beta-galactosidase, to peptides and galactoseand/or galacto-oligomers. These enzyme digestion products are moredigestible by the animal. Thus, hydrolases of the invention cancontribute to the available energy of the feed or food. Also, bycontributing to the degradation of galactan-comprising proteins, ahydrolase of the invention can improve the digestibility and uptake ofcarbohydrate and non-carbohydrate feed or food constituents such asprotein, fat and minerals.

In another aspect, hydrolase of the invention can be supplied byexpressing the enzymes directly in transgenic feed crops (as, e.g.,transgenic plants, seeds and the like), such as corn, soy bean, rapeseed, lupin and the like. As discussed above, the invention providestransgenic plants, plant parts and plant cells comprising a nucleic acidsequence encoding a polypeptide of the invention. In one aspect, thenucleic acid is expressed such that the hydrolase of the invention isproduced in recoverable quantities. The hydrolase can be recovered fromany plant or plant part. Alternatively, the plant or plant partcontaining the recombinant polypeptide can be used as such for improvingthe quality of a food or feed, e.g., improving nutritional value,palatability, and rheological properties, or to destroy an antinutritivefactor.

Paper or Pulp Treatment

The hydrolases of the invention can be in paper or pulp treatment orpaper deinking. For example, in one aspect, the invention provides apaper treatment process using hydrolases of the invention. In anotheraspect, paper components of recycled photocopied paper during chemicaland enzymatic deinking processes. In one aspect, hydrolases of theinvention can be used in combination with cellulases, pectate lyases orother enzymes. The paper can be treated by the following threeprocesses: 1) disintegration in the presence of hydrolases of theinvention, 2) disintegration with a deinking chemical and hydrolases ofthe invention, and/or 3) disintegration after soaking with hydrolases ofthe invention. The recycled paper treated with hydrolases can have ahigher brightness due to removal of toner particles as compared to thepaper treated with just cellulase. While the invention is not limited byany particular mechanism, the effect of hydrolases of the invention maybe due to its behavior as surface-active agents in pulp suspension.

The invention provides methods of treating paper and paper pulp usingone or more hydrolases of the invention. The hydrolases of the inventioncan be used in any paper- or pulp-treating method, which are well knownin the art, see, e.g., U.S. Pat. Nos. 6,241,849; 6,066,233; 5,582,681.For example, in one aspect, the invention provides a method for deinkingand decolorizing a printed paper containing a dye, comprising pulping aprinted paper to obtain a pulp slurry, and dislodging an ink from thepulp slurry in the presence of hydrolases of the invention (otherenzymes can also be added). In another aspect, the invention provides amethod for enhancing the freeness of pulp, e.g., pulp made fromsecondary fiber, by adding an enzymatic mixture comprising hydrolases ofthe invention (can also include other enzymes, e.g., pectate lyase,cellulase, amylase or glucoamylase enzymes) to the pulp and treatingunder conditions to cause a reaction to produce an enzymatically treatedpulp. The freeness of the enzymatically treated pulp is increased fromthe initial freeness of the secondary fiber pulp without a loss inbrightness.

Waste Treatment

The hydrolases of the invention can be used in a variety of otherindustrial applications, e.g., in waste treatment. For example, in oneaspect, the invention provides a solid waste digestion process usinghydrolases of the invention. The methods can comprise reducing the massand volume of substantially untreated solid waste. Solid waste can betreated with an enzymatic digestive process in the presence of anenzymatic solution (including hydrolases of the invention) at acontrolled temperature. This results in a reaction without appreciablebacterial fermentation from added microorganisms. The solid waste isconverted into a liquefied waste and any residual solid waste. Theresulting liquefied waste can be separated from said any residualsolidified waste. See e.g., U.S. Pat. No. 5,709,796.

In addition, the hydrolases (e.g., proteases) of the invention can beused in the animal rendering industry, to e.g., get rid of feathers,e.g., as described by Yamamura (2002) Biochem. Biophys. Res. Com.294:1138-1143. Alkaline proteases of the invention can also be used inthe production of proteinaceous fodder from waste feathers orkeratin-containing materials, e.g., as described by Gupta (2002) Appl.Microbiol. Biotechnol. 59:15-32.

Lubricants and Hydraulic Oils

The methods and compositions (enzymes of the invention, e.g., esterases,acylases, lipases, phospholipases or hydrolases of the invention) of theinvention can be used to prepare lubricants, such as hydraulic oils.Thus, the invention also provides lubricants and hydraulic oilscomprising a hydrolase of the invention. The purpose of a lubricant ofthe invention is to minimize friction and wear of metals. Lubricants canfurther comprise base fluids and additives improving the lubricativeproperties. See, e.g., U.S. Pat. No. 5,747,434.

Interesterification

In one aspect, the methods and compositions of the present invention canbe used to modify the properties of triglyceride mixtures, and, in oneaspect, their consistency. In one aspect, an enzyme of the invention canbe used in the presence of a catalyst such as sodium metal or sodiummethoxide to promote acyl migration between glyceride molecules suchthat the products consist of glyceride mixtures in which the fatty acylresidues are randomly distributed among the glyceride molecules.

In one aspect, the enzymes of the invention can be used to produceinteresterification products in the reaction where hydrolysis of fat isminimized so that lipase-catalyzed interesterification becomes thedominant reaction. These conditions may include, for example,restricting the amount of water in the system.

In one aspect, enzymes of the invention can be used to catalyzeinteresterification reaction using mixtures of triglycerides and freefatty acids, as described, e.g., in EP 0 093 602 B2. In these cases,free fatty acid can be exchanged with the acyl groups of thetriglycerides to produce new triglycerides enriched in the added fattyacid. In one aspect, 1,3-specific lipases of the invention can be usedto confine the reaction to the 1- and 3-positions of the glycerides,which allow to obtain a mixture of triglycerides unobtainable bychemical interesterification or reaction with a non-specific lipase. Inone aspect, non-specific lipases are used to attain results similar tochemical interesterification.

The ability to produce novel triglyceride mixtures using positionallyspecific lipases of the invention is useful to the oils and fatsindustry because some of these mixtures have valuable properties. Forexample, 1,3-specific lipase-catalyzed interesterification of1,3-dipalmitoyl-2-monoleine (POP), which is the major triglyceride ofthe mid-fraction of palm oil, with either stearic acid or tristearingives products enriched in the valuable1-palmitoyl-3-stearoyl-2-monoleine (POSt) and 1,3-distearoyl-2-monoleine(StOSt). POSt and StOSt are the important components of cocoa butter.Thus, one aspect of the invention provides an interesterificationreaction to produce cocoa butter equivalents from cheap startingmaterials.

In one aspect, the invention provides a method of production of a hardfat replacer using the 1,3-specific lipases of the invention. In oneaspect, a hard fat replacer comprises a mixture of palm mid-fraction andStOSt, POSt or StOSt/POSt of at least 85% purity.

Oral Care Products

The invention provides oral care product comprising hydrolases of theinvention. Exemplary oral care products include toothpastes, dentalcreams, gels or tooth powders, odontics, mouth washes, pre- or postbrushing rinse formulations, chewing gums, lozenges, or candy. See,e.g., U.S. Pat. No. 6,264,925.

Brewing and Fermenting

The invention provides methods of brewing (e.g., fermenting) beercomprising hydrolases of the invention. In one exemplary process,starch-containing raw materials are disintegrated and processed to forma malt. A hydrolase of the invention is used at any point in thefermentation process. For example, hydrolases (e.g., proteases) of theinvention can be used in the processing of barley malt. The major rawmaterial of beer brewing is barley malt. This can be a three stageprocess. First, the barley grain can be steeped to increase watercontent, e.g., to around about 40%. Second, the grain can be germinatedby incubation at 15 to 25° C. for 3 to 6 days when enzyme synthesis isstimulated under the control of gibberellins. In one aspect, hydrolasesof the invention are added at this (or any other) stage of the process.The action of hydrolases results in an increase in fermentable reducingsugars. This can be expressed as the diastatic power, DP, which can risefrom around 80 to 190 in 5 days at 12° C. Hydrolases (e.g., proteases)of the invention can be used in any beer or alcoholic beverage producingprocess, as described, e.g., in U.S. Pat. Nos. 5,762,991; 5,536,650;5,405,624; 5,021,246; 4,788,066.

Medical and Research Applications

Hydrolases, (e.g., proteases) of the invention can be used for cellisolation from tissue for cellular therapies in the same manner thatcollagenases. For example, metallo-endoproteinases and other enzymes ofthe invention that can cleave collagen into smaller peptide fragments,can be used as “liberase enzymes” for tissue dissociation and to improvethe health of isolated cells. “Liberase enzymes” can replace traditionalcollagenase. Proteases of the invention having collagenase I,collagenase II, clostripain and/or neutral protease activity can be usedfor tissue dissociation. In one aspect, for tissue dissociation,collagenase isoforms of the invention are blended with each other, and,optionally, with a neutral protease. In one aspect, the neutral proteaseis a neutral protease dispase and/or the neutral protease thermolysin.

Additionally, proteases of the invention can be used as antimicrobialagents, due to their bacteriolytic properties, as described, e.g., inLi, S. et. al. Bacteriolytic Activity and Specificity of Achromobacterb-Lytic Protease, J. Biochem. 124, 332-339 (1998).

Proteases of the invention can also be used therapeutically to cleaveand destroy specific proteins. Potential targets include toxin proteins,such as Anthrax, Clostridium botulinum, Ricin, and essential viral orcancer cell proteins.

Proteases of the invention can also be used in disinfectants, asdescribed, e.g., in J. Gen Microbiol (1991) 137(5): 1145-1153; Science(2001) 249:2170-2172.

Additional medical uses of the proteases of the invention include lipomaremoval, wound debraidment and scar prevention (collagenases), debridingchronic dermal ulcers and severely burned areas.

Enzymes of the invention (e.g., esterases, proteases, etc., of theinvention) can be used to in sterile enzymatic debriding compositions,e.g., ointments, in one aspect, containing about 250 collagenase unitsper gram. White petrolatum USP can be a carrier. In one aspect, enzymes(e.g., proteases) of the invention can be used in indications similar toSantyl® Ointment (BTC, Lynbrook, NY). Proteases of the invention canalso be used in alginate dressings, antimicrobial barrier dressings,burn dressings, compression bandages, diagnostic tools, gel dressings,hydro-selective dressings, hydrocellular (foam) dressings, hydrocolloidDressings, I.V dressings, incise drapes, low adherent dressings, odorabsorbing dressings, paste bandages, post operative dressings, scarmanagement, skin care, transparent film dressings and/or wound closure.Proteases of the invention can be used in wound cleansing, wound bedpreparation, to treat pressure ulcers, leg ulcers, burns, diabetic footulcers, scars, IV fixation, surgical wounds and minor wounds.

Additionally, enzymes of the invention can be used in proteomics and labwork in general. For instance, proteases can be used in the same manneras DNA restriction enzymes.

Other Industrial Applications

The invention also includes a method of increasing the flow ofproduction fluids from a subterranean formation by removing a viscous,protein-containing, damaging fluid formed during production operationsand found within the subterranean formation which surrounds a completedwell bore comprising allowing production fluids to flow from the wellbore; reducing the flow of production fluids from the formation belowexpected flow rates; formulating an enzyme treatment (comprising anenzyme of the invention) by blending together an aqueous fluid and apolypeptide of the invention; pumping the enzyme treatment to a desiredlocation within the well bore; allowing the enzyme treatment to degradethe viscous, protein-containing, damaging fluid, whereby the fluid canbe removed from the subterranean formation to the well surface; andwherein the enzyme treatment is effective to attack protein in cellwalls.

Hydrolases of the invention can be used for peptide synthesis, in theleather industry, e.g., for hide processing, e.g., in hair removaland/or bating, for waste management, e.g., removal of hair from drains,in the photography industry, e.g., for silver recovery from film, in themedical industry, e.g., as discussed above, e.g., for treatment ofburns, wounds, carbuncles, furuncles and deep abscesses or to dissolveblood clots by dissolving fibrin, for silk degumming.

In other aspects, enzymes of the invention can be used as flavorenhancers in, for example, cheese and pet food, as described, e.g., inPommer, K., Investigating the impact of enzymes on pet foodpalatability, Petfood Industry, May 2002, 10-11.

In yet another embodiment of the invention, enzymes of the invention canbe used to increase starch yield from corn wet milling, as described,e.g., in Johnston, D. B., and Singh, V. Use of proteases to Reduce SteepTime and SO₂ requirements in a corn wet-milling process, Cereal Chem.78(4):405-411.

In other aspects, enzymes of the invention can be used in biodefense(e.g., destruction of spores or bacteria). Use of enzymes in biodefenseapplications offer a significant benefit, in that they can be veryrapidly developed against any currently unknown biological warfareagents of the future. In addition, proteases of the invention can beused for decontamination of affected environments.

Additionally, enzymes of the invention can be used in biofilmdegradation, in biomass conversion to ethanol, and/or in the personalcare and cosmetics industry.

Enzymes of the invention can also be used to enhance enantioselectivity,as described, e.g., in Arisawa, A. et. al. Streptomyces Serine Protease(DHP-A) as a New Biocatalyst Capable of Forming Chiral Intermediates of1,4-Diohydropyridine Calcium Antagonists. Appl Environ Mircrobiol 2002June; 68(6):2716-2725; Haring, D. et. al. Semisynthetic Enzymes inAsymmetric Synthesis:Enantioselective Reduction of RacemicHydroperoxides Catalyzed by Seleno-Subtilisin. J. Org. Chem. 1999,64:832-835.

EXAMPLES Example 1 Exemplary Lipase Assays

The following example describes exemplary assays to screen for lipaseactivity. In one aspect, these exemplary assays can be used as routinescreens to determine if a polypeptide is within the scope of theinvention. Such assays include use of pH indicator compounds to detectcleavage of fatty acids from triglycerides, spectrophotometric methods,HPLC, GC, MS, TLC and others. Jaeger (1994) FEMS Microbiol. Rev.15:29-63; Ader (1997) Methods Enzymol. 286:351-386; Vorderwülbecke(1992) Enzyme Microb. Technol. 14:631-639; Renard (1987) Lipids 22:539-541.

In one aspect, the methods of the invention screen for regio-selectivelipases, e.g., Sn-1, Sn-3 and/or Sn-3 regio-selective lipases. In oneaspect, the substrates 1,3-diamide and 1,3-diether TAG analogues areused to target, or select for, Sn-2 selective lipases. In one aspect,the methods of the invention screen for lipases that exhibitregioselectivity for the 2-position of lipids, e.g., TAGs. Structuredsynthesis of lipids using Sn-2 selective lipases can be useful for thesynthesis of a variety of TAGs, including 1,3-diacylglycerides(1,3-DAGs) and components of cocoa butter.

In one aspect, regio-selective lipases, including Sn-2 selectivelipases, are characterized for regioselectivity using rigorousanalytical methods. This can eliminate false results due to acylmigration. In one aspect, lipases are tested for Sn2 specificity usinganalytical methods such as NMR spectroscopy. Also, a structuredtriacylglyceride of the ABA-type (where A and B denote fatty acidsdistributed along the glycerol backbone) can be subjected to hydrolysisor alcoholysis using lipases (e.g. of the invention) followed byanalysis of the partial glycerides and fatty acid (esters) formed.Alcoholysis conditions at controlled water activity, e.g. using primaryalcohols such as methanol or ethanol are preferred as undesired acylmigration can be avoided need different references. Sn-2 selectivity oflipases was reported, however, the extent of sn-2 selectivity was verylow (Briand (1995) Eur. J. Biochem. 228: 169-175; Rogalska (1993)Chirality 5, 24-30.

In one aspect, regio-selective lipases are assayed for theirregio-specificity (Sn2 versus Sn1/Sn3 versus Sn1,3) on appropriatelipids, such as tripalmitin, tristearin, triolein, tricaprylin, andtrilaurin, and also for their fatty acid specificity.

Screening for Lipase/Esterase Activity

Colonies are picked with sterile toothpicks and used to singly inoculateeach of the wells of 96-well microtiter plates. The wells contained 250μL of LB media with 100 μg/mL ampicillin, 80 μg/mL methicillin, and 10%v/v glycerol (LB Amp/Meth, glycerol). The cells were grown overnight at37° C. without shaking. This constituted generation of the “SourceGenBank.” Each well of the Source GenBank thus contained a stock cultureof E. coli cells, each of which contained a pBluescript with a uniqueDNA insert.

Plates of the source GenBank were used to multiply inoculate a singleplate (the “condensed plate”) containing in each well 200 μL of LBAmp/Meth, glycerol. This step was performed using the High DensityReplicating Tool (HDRT) of the Beckman Biomek with a 1% bleach, water,isopropanol, air-dry sterilization cycle in between each inoculation.Each well of the condensed plate thus contained 10 to 12 differentpBluescript clones from each of the source library plates. The condensedplate was grown for 16 hours at 37° C. and then used to inoculate twowhite 96-well Polyfiltronics microtiter daughter plates containing ineach well 250 μL of LB Amp/Meth (no glycerol). The original condensedplate was put in storage −80° C. The two condensed daughter plates wereincubated at 37° C. for 18 hours.

The short chain esterase ‘600 μM substrate stock solution’ was preparedas follows: 25 mg of each of the following compounds was dissolved inthe appropriate volume of DMSO to yield a 25.2 mM solution. Thecompounds used were 4-methylumbelliferyl proprionoate,4-methylumbelliferyl butyrate, and 4-methylumbelliferyl heptanoate. Twohundred fifty microliters of each DMSO solution was added to ca 9 mL of50 mM, pH 7.5 HEPES buffer which contained 0.6% of Triton X-100 and 0.6mg per mL of dodecyl maltoside (Anatrace, Maumee, Ohio). The volume wastaken to 10.5 mL with the above HEPES buffer to yield a slightly cloudysuspension.

The long chain ‘600 μM substrate stock solution’ was prepared asfollows: 25 mg of each of the following compounds was dissolved in DMSOto 25.2 mM as above. The compounds used were 4-methylumbelliferylelaidate, 4-methylumbelliferyl palmitate, 4-methylumbelliferyl oleate,and 4-methylumbelliferyl stearate. All required brief warming in a 70°C. bath to achieve dissolution. Two hundred fifty microliters of eachDMSO solution was added to the HEPES buffer and diluted to 10.5 mL asabove. All seven umbelliferones were obtained from Sigma Chemical Co.(St. Louis, Mo.).

Fifty μL of the long chain esterase or short chain esterase ‘600 μMsubstrate stock solution’ was added to each of the wells of a whitecondensed plate using the Biomek to yield a final concentration ofsubstrate of about 100 μM. The fluorescence values were recorded(excitation=326 nm, emission=450 nm) on a plate-reading fluorometerimmediately after addition of the substrate. The plate was incubated at70° C. for 60 minutes in the case of the long chain substrates, and 30minutes at RT in the case of the short chain substrates. Thefluorescence values were recorded again. The initial and finalfluorescence values were compared to determine if an active clone waspresent.

To isolate the individual clone which carried the activity, the SourceGenBank plates were thawed and the individual wells used to singlyinoculate a new plate containing LB Amp/Meth. As above, the plate wasincubated at 37° C. to grow the cells, 50 μL of 600 μM substrate stocksolution was added using the Biomek and the fluorescence was determined.Once the active well from the source plate was identified, cells fromthis active well were streaked on agar with LB/Amp/Meth and grownovernight at 37° C. to obtain single colonies. Eight single colonieswere picked with a sterile toothpick and used to singly inoculate thewells of a 96-well microtiter plate. The wells contained 250 μL of LBAmp/Meth. The cells were grown overnight at 37° C. without shaking. A200 μL aliquot was removed from each well and assayed with theappropriate long or short chain substrates as above. The most activeclone was identified and the remaining 50 μL of culture was used tostreak an agar plate with LB/Amp/Meth. Eight single colonies werepicked, grown and assayed as above. The most active clone was used toinoculate 3 mL cultures of LB/Amp/Meth, which were grown overnight. Theplasmid DNA was isolated from the cultures and utilized for sequencing.

Example 2 Exemplary Structured Lipid Synthesis Methods

The following example describes exemplary structured lipid synthesismethods of the invention using lipases of the invention.

In one aspect, the invention provides a “Forced Migration Methodology”for the structured synthesis of lipids using the lipases of theinvention. This method provides for the efficient synthesis of a varietyof structured lipids, including 1,3-DAGs and components of cocoa butter,as illustrated in FIG. 7. In one aspect, the method for producingstructured lipids, in this example a structured triacylglyceride (sTAG),comprises three major steps:

-   -   1. Regiospecific hydrolysis or alcoholysis (e.g. ethanolysis) of        a TAG using a Sn1-specific or Sn3-specific lipases to yield a        2,3 or 1,2-DAG, respectively;    -   2. Promotion of acyl migration in a purified or unpurified DAG        under kinetically-controlled conditions using ion-exchange        resins or other method(s), resulting in the structured 1,3-DAG;        and    -   3. Fatty acid-specific lipase catalyzed addition of a fatty acid        or a fatty acid derivative, such as a fatty acid ethyl ester or        vinyl ester, at the Sn2 position, yielding the sTAG.

This route can provide access to two target groups of lipids, 1,3-DAGsand sTAGs with the same set of enzymes and methodology. The method canuse lipases that have Sn1 and/or Sn3 regiospecificity; such enzymes arecommercially available (Rhizopus delemar (Amano, Japan) and Rhizomucormiehei (Novozymes, Denmark

In one aspect, Rhizopus sp. lipases and Rhizomucor miehei lipases areused. These lipases are known to exhibit higher specificity forhydrolysis of fatty acids in the Sn1 position compared with the Sn3position. In one aspect, the methods further comprise use of theseRhizopus and Rhizomucor meihei lipases to confirm Step 1, of FIG. 7,i.e., the regiospecific hydrolysis or alcoholysis (e.g. ethanolysis) ofa TAG using an Sn1-specific or Sn3-specific lipase to yield a 2,3 or1,2-DAG, respectively.

In one aspect, the invention provides a forced migration methodsupplemented with glycerol. Addition of glycerol to the enzyme reactionprior to the treatment with anion exchange resin (or other migrationcatalysts) can be a way to increase yields of 1,3 DAGs in forcedmigration reactions, as illustrated in FIG. 27.

In one aspect, the methods further comprise confirmation of Step 2, FIG.7, by treatment of purified or unpurified 1,2-DAG or 2,3-DAG with anion-exchange resin, for example, an ion-exchange column, under a varietyof conditions. Major variables include the nature of the ion-exchangeresin, pH, flow rates, buffer type and ionic strengths. Alternativemethods of promoting acyl migration can be used. In one aspect, the acylmigration can be performed under non-equilibrium conditions, e.g., suchthat the end product contains greater ratios of one product overanother, for example, such that the end product contains a 2:1 ratio of1,3-DAG to 2,3-DAG. Substrates for the Step 2 validation studies areavailable commercially.

In one aspect, acyl migration in 2,3-DAGs (or 1,2-DAGs) is promotedunder kinetic conditions such that the final product is a purified1,3-DAG in greater than about a 70% yield.

In one aspect, Step 3, FIG. 7, uses a lipase that is fatty acidspecific, but there are no regiospecific requirements given a pure1,3-DAG as substrate. The lipase from Geotrichium candidum exhibits avery high specificity for fatty acids that have 9 unsaturation. Thisenzyme is readily available and can be utilized to confirm Step 3.

Example 3 Exemplary Structured Synthesis of Cocoa Butter Alternatives(CBAs)

The following example describes exemplary structured lipid synthesismethods of the invention using the lipases of the invention. Thisexample describes the structured synthesis of triacylglycerides (sTAGs)as cocoa butter alternatives (CBAs).

Natural cocoa butter consists mostly of three TAG:1,3-dipalmitoyl-2-oleoylglycerol (POP), 1,3-distearoyl-2-oleoylglycerol(SOS) and 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS). The relativeproportions of these three TAGs differ somewhat depending upon thesource of cocoa butter, but are approximately 21:31:48 (POP:SOS:POS).The methods of the invention provide for the structured synthesis of acocoa butter alternatives having any proportion of POP:SOS:POS,including the natural 21:31:48 POP:SOS:POS. The methods of the inventionprovide for the structured synthesis related TAGs, e.g.,1-oleoyl-2,3-dimyristoylglycerol (OMM). The methods of the inventionalso provide for the selective processing of natural cocoa butter usingthe lipases of the invention.

In one aspect, both the Sn2 lipase and the methods outlined above(including Example 2) are used in the synthesis of key structured lipidsof CBAs. Lipases can be assayed for their regiospecificity (Sn2 versusSn1/Sn3 versus Sn1,3) on appropriate lipids, such as tripalmitin,tristearin, triolein, tricaprolein, and trilaurein. Lipases also can beassayed for their fatty acid specificity.

Example 4 Exemplary Structured Synthesis of Nutraceuticals

The following example describes exemplary structured lipid synthesismethods of the invention for making nutraceuticals using the lipases ofthe invention.

In one aspect, 1,3-DAGs are synthesized for use in nutraceuticals.1,3-DAGs are the products of the first step of the Sn2 lipase synthesisof sTAGs shown in FIG. 6 and first two steps of the synthesis ofstructured lipids as shown in FIG. 7 and FIG. 8. Routine assaying oflipases for Sn2 versus Sn1 or Sn3 specificity can provide the data todetermine the optimum lipases and methodology for different 1,3-DAG andnutraceutical synthesis applications.

In one aspect, poly-unsaturated fatty acids (PUFAs) are made using themethods and lipases of the invention, e.g., using a protocol as setforth in FIG. 9, bottom. PUFAs are themselves valuable commodities andcan be extracted from PUFA-containing fat sources, such as fish oil,using PUFA-specific lipases of the invention. In one aspect, theinvention uses enzymes that can distinguish between esters of differentPUFAs, e.g. docosahexaenoic acid (DHA) versus eicosapentaenoic acid(EPA), facilitating the development of highly purified products (S.Wongsakul et al., Eur. J. Lipid Sci. Technol. 105 (2003) 68-73). Lipasescan be tested for their specificities on a variety of PUFA esters andglycerol esters.

Example 5 Exemplary Structured Synthesis of Lipids ContainingPoly-Unsaturated Fatty Acids

The following example describes exemplary structured lipid synthesismethods of the invention for making lipids containing poly-unsaturatedfatty acids (PUFAs) using the lipases of the invention. ThesePUFA-containing lipids can be used in foods, feeds, cosmetics,pharmaceuticals and drug delivery agents, nutraceuticals and the like.

In one aspect, fish oil is a starting material, since in fish oil themajority of fatty acids at the 2 position are PUFAs. In one aspect, themethods comprise a 1,3-lipase-catalyzed interesterification of fish oilwith medium-chain fatty acid esters to form MLM-type lipids(triacylglycerols (TAG) can be of types MML, MLM, MLL, and LML (M,medium-chain fatty acid; L, long-chain fatty acid, see, e.g., Kurvinen(2001) Lipids 36:1377-1382)).

In one aspect, the invention provides methods for making PUPA-containingsTAGs, as illustrated in FIG. 9A, and 2-PUFA sMAGs and purified PUPAs,as illustrated in FIG. 9B. Any appropriate starting oil can be used. Inone aspect, if it is more economic to use fish-oil fatty acids

instead of purified PUPAs, lipases with specificity for fatty acid B (inFIG. 9A) and PUFAs can be used. Lipases can be screened for fatty acidspecificity on simple esters and glycerol esters. In one aspect, 2-PUFAMAGs are synthesized from fish oil using either a Sn1,3-lipase (see FIG.9B) or a non-regiospecific lipase that does not cleave PUPA esters.

Example 6 Exemplary Growth-Kill Assay

The following example describes an exemplary Growth-Kill assay fortesting the activity of the lipases of the invention. See, e.g., Chem.Communication (2002) 1428-1429.

The Growth-Kill assay provides a method for in vivo selection ofenzymes, e.g. lipases, or mutants thereof, with desirable properties.The assay combines two components, a growth component and a killcomponent. The first of these is a substrate from which the enzyme, e.g.lipase, liberates an element which allows the host organisms to grow,e.g. a carbon source. The second of these is a substrate from which theenzyme, e.g. lipase, liberates an element which prevents the hostorganism from growing or kills the host organism, e.g. an antibiotic.

The invention provides methods of modifying a nucleic acid encoding alipase to generate an enzyme with modified properties. A Growth-Killassay can be used to discriminate between two fatty acids, to determineif a lipase has altered enzyme specificity, to determine if a lipase iswithin the scope of the invention, and the like. An exemplaryGrowth-Kill assay is outlined in FIG. 11, where R1 is a growth substratefor the screening host and R2 is a substance that is toxic to the celland kills the host if released from the ester. In one aspect, growthsubstrates (1-acyl glycerol esters) and kill substrates (3-acylchloramphenicol esters) are used

Example 7 Protocol for the Synthesis of 1,3-Diglyceride

The following example describes an exemplary protocol used to practicethe compositions (the lipases) and the methods of the invention. Theseexemplary protocols can also be used to determine if a lipase is withinthe scope of the invention. In one aspect, an exemplary protocol for thesynthesis of 1,3-diglyceride (1,3-DG) and structured lipids usinglipases of the invention is described.

In one aspect, glycerol and free fatty acid (FFA) or fatty acid vinylester (FAVE) are esterified immobilized glycerol (see, e.g., J. Am. OilChem. Soc., 1992, 69:955-960). The immobilized glycerol can be on asilica gel. In one exemplary assay, Lipozyme RM IM™ (an immobilized1,3-specific lipase, Novozymes, Denmark), with MTBE, at room temperaturewas used. This has the advantage of high yield and purity of 1,3-DG, andfast reaction. However, MTBE is not allowed in food use and there isdifficulty in separation of immobilized enzyme and silica gel

In one aspect, the esterification is done using non-immobilizedglycerol. In one exemplary assay, esterification of glycerol and fattyacid (FFA) or fatty acid vinyl ester (FAVE) is in a solvent-free/organicsolvent with an immobilized lipase from Candida antarctica type B(CAL-B), a lipase from Rhizopus delemar immobilized on EP100 (D-EP100),and Lipozyme RM IM™ (Novozymes, Denmark), at 0° C. or room temperature(RT). One advantage of this exemplary protocol is the solvent-freecondition; it allows ease of separation of immobilized enzyme and with afurther purification step, a moderate-high yield.

In one aspect, alcoholysis and hydrolysis of triglycerides (TG) isaccomplished using 1,3-regiospecific lipases, D-EP100 and Lipozyme RMIM™ (Novozymes, Denmark), organic solvents, preferentially at controlledwater activity. In this reaction, the substrates (natural oil) are cheapand the reaction provides an acceptable yield. Most DAG formed is1,2(2,3)-DAG.

In one aspect, an exemplary reaction involved induction of acylmigration of 1,2(2,3)-DAG. Most DAG obtained from alcoholysis andhydrolysis of TAG is 1,2 (2,3)-DaG. It is thus necessary to induce theacyl migration of 1,2(2,3)-DAG to 1,3-DAG. Several factors were studied,including ion-exchangers, acid or base, heat, carrier, water activity.One exemplary reaction involved the esterification between 1,3-DAG andFFA or FAVE using an sn2-specific enzyme or a non-regiospecific lipasebut specific to fatty acid change length or specific fatty acid.

Esterification Using Immobilized Glycerol)

1,3-DAG was synthesized from glycerol, 1 mmol, immobilized on 4 g silicagel) and vinyl laurate (2 mmol) in 8 ml methyl-tert-butyl ether (MTBE)at room temperature using Lipozyme RM IM™ (Novozymes, Denmark) (10%based on glycerol weight) as catalyst (Matthias et al. 1992???). Thereaction was carried out in a 10-ml vial and the reaction mixture wasmixed by magnetic stirrer (500 rpm). After 24 hours (h), enzyme wasseparated from the reaction mixture by filtration to stop the reaction.The filtrate was evaporated under vacuum. 1,3-DAG in oily residue wasrecovered and purified by crystallization in dry methanol at 4° C.followed by filtration. See, e.g., J. Am. Oil Chem. Soc., 1992,69:955-960).

Esterification Using Non-Immobilized Glycerol

1,3-DAG was synthesized by esterification of glycerol (lmmol) and FFA orFAVE (2 mmol) in a solvent-free condition or in organic solvent at 0° C.using CAL-B (10% based on glycerol weight) as catalyst. The reaction wascarried out in a 4-ml vial and the reaction mixture was mixed bymagnetic stirrer (400 rpm). Activiated molecular sieve was added in areaction with FFA to remove produced water from the reaction mixture. Insome reactions organic solvent (2 ml) was added to the reaction mixtureto dissolve a solid FFA. The reaction was stopped by dissolving thereaction mixture in n-hexane (in case of a solvent-free reactioncondition) and centrifuged to separate immobilized enzyme from thereaction mixture. The 1,3-DAG was recovered and purified bycrystallization at −20° C. If high contents of MAG were present, arecrystallization in dry methanol at −20° C. afforded pure 1,3-DAG.Samples (10 μl) were periodically withdrawn during the reaction todetermine the acylglycerol composition. Samples were pretreated beforeanalysis by adding 0.3 ml Folsh's solution (chloroform:methanol, 2:1 byvol) and 0.3 ml distilled water, mixed for 30 sec, followed bycentrifuging (10000 rpm, 2 min) Organic layer was used for analysis byIATROSCAN™ (Shell-usa, Fredericksburg Va.).

Alcoholysis and Hydrolysis of TAG

TAG (3 mmol) was dissolved in organic solvent (2 ml) andpre-equilibrated over a saturated-salt solution at a_(W) 0.11 for 48 h(only for alcoholysis reaction). Dry ethanol or water (3 mmol) was addedand the reaction mixture was incubated at 40° C. for 15 min Immobilizedlipase (10% based on TAG weight) was added to start the reaction. Thereaction was carried out in a 4-ml screw-capped vial and the reactionmixture was mixed by magnetic stirrer (400 rpm). An aliquot of thereaction mixture was periodically withdrawn and diluted with chloroformto appropriate dilution, followed by analysis with IATROSCAN™(Shell-usa, Fredericksburg Va.) to determine acylglycerol compositionImmobilized lipase was separated from the reaction mixture after 48 h bycentrifugation to stop the reaction.

Induction of Acyl Migration

Effect of temperature, FFA (oleic acid), carrier (celite) andion-exchanger on acyl migration of 1,2-dipalmitin (1,2-DP, this alsoincludes the stereoisomer 2,3-DP) were studied. 1,2-DP was dissolved inn-hexane (8 mg/ml). Oleic acid (2-4 mmol) or celite (8 mg) orion-exchanger (10-100 mg) was added directly to the reaction mixture.All reactions were carried out in a 1.5-ml Eppendorf reaction vial withshaking (1400 rpm) at room temperature (25° C.), except when testingeffect of temperature, then the reaction was carried out at 40 or 60° C.

Synthesis of Structured Triglycerides (ST) from 1,3-Diglycerides(1,3-DAG) and Free Fatty Acid or Fatty Acid Vinyl Este

Structured triglycerides (ST) was synthesized by esterification of1,3-DAG and oleic acid (OA) or oleic acid vinyl ester (OAVE) in n-hexaneat 60° C. using immobilized lipase from Pseudomonas sp. (Amano PS-D,Amano Enzyme USA, Elgin, Ill.) as biocatalyst. 0.1 mmol of 1,3-DG (45.7mg of 1,3-dilaurin or 34.4 mg of 1,3-dicaprylin) and 0.2 mmol of OA(28.2 mg) or OAVE (60.2 mg) was dissolved in 1 ml n-hexane in a 2-mlscrew-capped vial. Activated molecular sieve was added when OA was usedas acyl donor. The reaction was started by addition of PS-D (10% weightof 1,3-DG). The vials were shaken at 1400 rpm at 60° C. An aliquot ofreaction mixtures was withdrawn for analysis with IATROSCAN™ (Shell-usa,Fredericksburg Va.). ST thus obtained was purified on TLC plate and theTAG band was scrapped of and methylated followed by GC analysis.

Acylglycerol composition was determined by IATROSCAN™ (Shell-usa,Fredericksburg Va.) analysis (TLC-FID). Total fatty acid composition ofABA-ST was determined by GC analysis of corresponding methylesters.Purity of 1,3-DAG was confirmed by ¹H-NMR spectroscopy.

Determination of Fatty Acid Composition by GC Analysis

10 mg of 1,3-DG was methylated with 0.5% NaOH in methanol (500 μl) andthen incubated for 10 min at 60° C. The methylesters were extracted withn-hexane (400 μl) for 1 min. The n-hexane layer was washed with 200 μldistilled water and dried over anhydrous sodium sulfate. Analysis wascarried out with a Hewlett-Packard 5890 (series II) gas chromatograph(GS) (Hewlett-Packard, USA) on a FFAP column (Permabond FFAP-DF-0.25, 25m×0.25 mm i.d., Macherey-Nagel GmbH, Düren, Germany). Hydrogen was usedas the carrier gas. The temperature program used was 150° C. (4° C./min,0.50 min), 170° C. (5° C./min), 195° C. (10° C./min) and 215° C. (9.50min). Injector and detector temperatures were 250° C. Response factorswere determined using a standard mixture of fatty acid methylesters

Determination of Glyceride Composition by TLC/FID Analysis

Changes in glyceride composition during reaction were quantitativelydetermined using Iatroscan analytical method. Before analysis, a blankof the chromarod was scanned. After treating chromarod with boric acid(3%) and drying for 5 min, 1 μl of the reaction medium (diluted inchloroform at appropriate dilution) is spotted onto the chromarod andthe spotted sample was developed for 10 cm in a mixture ofbenzene:chloroform:acetic acid (50:30:0.5, by vol). After drying, thechromarod in an oven at 110° C. for 5 min, scanning is performed at ahydrogen flow rate of 160 ml/min and an air flow rate of 2.0 l/min toproduce a chromatogram.

HPLC Separation of Triacylglycerols

The composition of the triacylglycerols formed during the enzymaticesterification was characterized by HPLC using a nucleosil C₁₈ column,(5 μm, 250×4 mm, Sykam, Gilching, Germany) and an evaporative lightscattering detector (ELSD) (Polymer labs) at a flow rate of 1.5 ml/min.The purpose of ELSD is to complement ultraviolet (UV) detection ofsolutes, and to detect solutes, which do not absorb UV light such asmedium-chain triglycerides. The principle of ELSD applies to all soluteshaving a lower volatility than the mobile phase. Elution was performedusing a gradient elution system of acetonitrile and dichloromethane (70%to 55% acetonitrile over 10 min, followed by 55% to 70% acetonitrileover 8 minute).

Regiospecific Analysis of Triglycerides

The regiospecific analysis of oil was conducted by Grignard degradationwith allylmagnesium bromide followed by gas chromatograph (GS) analysis.20 mg of TG was dissolved in dry diethyl ether (2 ml). 800 μl of allylmagnesium bromide solution (1M) was added, and the mixture was shakenfor 30 second, then 300 μl glacial acetic acid was added, followed by 5ml of 0.4-M boric acid to stop the reaction. A mixture of deacylatedproducts was extracted with diethyl ether. This extract was washed with5 ml solution of aqueous boric acid (0.4 M)/aqueous NaHCO₃ (2%), 50:50(vol/vol). The ether layer was directly subjected to TLC plate, whichwas impregnated with boric acid, to isolate each fraction of deacylatedproducts. The plate was developed with a chloroform/acetone/acetic acidsolution (85:15:1, by vol) as developing system. The 1-MG bands werescraped off and methylated to determine their fatty acid compositionusing the same method described above. The molar percentage of fattyacid composition at sn 1(3)- and sn 2-positions of the produced TG werecalculated. Equation for % FA in sn2-position is shown below:

[% FA _(sn2-position)]=3[% FA _(TG)]−2[% FA _(1-MG)]

where [% FA_(1-MG)] and [% FA_(TG)] indicated for each fatty acid, itspercentage found in 1-monoglyceride and in triglycerides, respectively.

Dicaprylin (1,3-DCy)

1,3DCy was purified by crystallization from n-hexane at −20° C. severaltimes. When high amount of MG was presented, the second crystallizationin dry methanol has to be done to obtain 1,3-DCy in high purity (>98%).Highest yield of 1,3-DCy (93%) obtained from esterification betweenvinyl caprylate (CyVE) and glycerol at 0° C. in a solvent-free conditioncatalyzed by CAL-B. The yield thus obtained was higher than the yieldobtained (75%) in literature (see, e.g., J. Am. Oil Chem. Soc., 1992,69:955-960), see FIG. 12. FIG. 12 illustrates data of variousesterification reactions in the synthesis of 1,3-DCy. Method 1 is theesterification of glycerol and caprylic acid in a solvent-free conditionat 0° C. catalyzed by CAL-B. Method 2 is the esterification of glyceroland caprylic acid vinyl ester in a solvent-free condition at 0° C.catalyzed by CAL-B. Method 3 see, e.g., J. Am. Oil Chem. Soc., 1992,69:955-960) is the esterification of glycerol immobilized on silica geland caprylic acid vinyl ester in MTBE at room temperature catalyzed byLipozyme RM IM™ (Novozymes, Denmark). DG=1,3-dicaprylin,MG=1-monocaprylin.

Esterification between caprylic acid (Cy) and glycerol gave moderateyield (55%) at lower reaction rates and high amount of MAG was producedduring the reaction. The yield could be increased up to 65% byincreasing the reaction temperature from 0° C. to room temperature.CAL-B gave higher yield and less MAG and allowed faster reaction ratethan Lipozyme RM IM™ (Novozymes, Denmark) in esterification of glyceroland Cy or CyVE, both in organic solvent and a solvent-free condition.

Studies on effect of ratio of glycerol:caprylic acid on esterificationreaction showed that initial reaction rate decreased and the yield of1,3-DCy slightly increased with increasing ratio from 1:2 to 1:6, asillustrated in (FIG. 13). FIG. 13 summarizes data showing the effect ofsubstrate ratio on esterification between glycerol and caprylic acid inn-hexane at 0° C. catalyzed by CAL-B (DG=1,3-dicaprylin,MG=1-monocaprylin).

1,3-Dilaurin (1,3-DLa)

1,3DLa was easily purified and recovered by crystallization in drymethanol at room temperature (RT) or in hexane at −20° C. (purity >98%).When lauric acid (La) was used as an acyl-donor, solvent was added todissolve the FFA. Though the method as described in J. Am. Oil Chem.Soc., 1992, 69:955-960,allowed faster reaction rate with high yield of1,3-DLa (65%), the highest yield (78%) of 1,3-DLa obtained fromesterification between glycerol and lauric acid vinyl ester (LaVE) at 0°C. in a solvent-free condition catalyzed by CAL-B after 24 h, asillustrated in FIG. 14. FIG. 14 summarizes data of various synthesis of1,3-dilaurin. Method1=esterification of glycerol and lauric acid inn-hexane at 0° C. catalyzed by CAL-B; Method2=esterification of glyceroland lauric acid vinyl ester in a solvent-free condition at 0° C.catalyzed by CAL-B; Method3 (Schneider's method)=esterification ofglycerol (immobilized on silica gel) and lauric acid vinyl ester in MTBEat room temperature catalyzed by Lipozyme RM IM™ (Eurzyme, Dublin,Ireland). (DG=1,3-dilaurin, MG=1-monolaurin). LaVE, as acyl donor,allowed higher yield and faster reaction with less amount of MG than La.

CAL-B gave highest yield and fastest reaction rate of esterification ofLaVE and glycerol at 0° C., while Lipozyme RM IM™ gave moderate yield of1,3-DLa with high amount of MG and Lipozyme TL™ showed very low activityin the same reaction condition. When increasing reaction temperature to25° C., Lipozyme RM IM™ showed higher activity, while CAL-B was lessactive. In the esterification reaction between glycerol and lauric acidin n-hexane catalyzed by CAL-B, reaction rate and the yield of 1,3-DLadecreased with increasing amount of La, as shown in FIG. 15. FIG. 15summarizes the effect of substrate ration o esterification of glyceroland lauric acid in n-hexane at room temperature catalyzed by CAL-B(DG=1,3-dilaurin, MG=1-monolaurin).

1,3-Dipalmitin (1,3-DP) and 1,3-distearin (1,3-DS)

Reactions were carried out in organic solvent and at higher temperature(25° C. to 40° C.) due to a low solubility of palmitic acid (PA) andstearic acid (SA). The DG yield was lower than the yield of the reactionwith La or Cy. Highest yield (80%) and fastest reaction rate wasobtained from esterification of glycerol and palmitic acid vinyl ester(PAVE) in MTBE at 40° C. catalyzed by D-EP100. Reaction reached theequilibrium within 6-8 h with low amount of MG. Esterification of PA andimmobilized glycerol gave higher yield and faster reaction thanesterification with free glycerol, as shown in FIG. 16. FIG. 16summarizes the synthesis of 1,3-dipalmitin. Method1=esterification ofglycerol and palmitic acid in MTBE at 40° C. catalyzed by D-EP100;Method2=esterification of glycerol and palmitic acid vinyl ester in MTBEat 40° C. catalyzed by D-EP100; Method 3 (Schneider'smethod)=esterification of glycerol (immobilized on silica gel) andpalmitic acid vinyl ester in MTBE at room temperature catalyzed byLipozyme RM IM™. (DG=1,3-dipalmitin, MG=1-monopalmitin).

D-EP100 gave higher yield and activity than Lipozyme RM IM™ in all casesof 1,3-DP synthesis. Moreover, Lipozyme RM IM™ showed no activity inesterification of PA or PAVE when the reaction was performed in MTBE andlow activity in n-hexane. D-EP100 preferred the esterification of PAthan SA, while not much different activity on PA and SA was observedwith Lipozyme RM IM™, as illustrated in FIG. 17. FIG. 17 summarizes datafor the esterification of glycerol and palmitic (C16:O) or stearic(C18:0) acid in n-hexane at 40° C. (RM=Lipozyme RM IM™, DEP=D-EP100,DG=1,3-diglycerides, MG=1-monoglycerides).

Alcoholysis of Triglycerides

Alcoholysis of pure triglycerides (TGs), including trilaurin,tripalmitin and tristearin, was carried out. Most diglyceride (DG)obtained from alcoholysis reaction were 1,2-DG. A high amount ofunreacted TG remained in the reaction mixture. Acyl migration wasobserved during the reaction, especially in hydrolysis reaction, asillustrated in FIG. 18. FIG. 18 shows data from alcoholysis reactionshowing the 1,3-DS/1,2-DS ratio during alcoholysis and hydrolysis oftristearin. DEP=D-EP100, RM=Lipozyme RM IM, Hx=n-hexane, HYD=hydrolysis,ALC=alcoholysis.

The reaction catalyzed by Lipozyme RM IM™, though gave lower yield,showed higher acyl migration than the reaction catalyzed by D-EP100. Lowacyl migration was observed in alcoholysis using CAL-B after 6 h. Thiscould be because 1,2-DG and 1,3-DG were produced at the same timeaccording to the non-specificity of CAL-B.

D-EP100 showed higher activity than CAL-B and Lipozyme RM, respectively,in alcoholysis of tripalmitin and tristearin. Effect of a_(W:) Higheryield and less MG was obtained from alcoholysis at aW 0.11 than 0.43. Itwas found that MG was increased with increasing a_(W). Effect ofsolvents (on yield and acyl migration): Highest yield was obtained withMTBE. Alcoholysis in n-hexane and isooctane gave moderate yield, whileacetone was a poor solvent for Lipozyme RM. The reaction performed inn-hexane showed faster acyl migration than in MTBE, as illustrated inFIGS. 18 and 19. FIG. 19 illustrates data from the hydrolysis oftrilaurin at 60° C. by Lipozyme RM IM™ (DG=dilaurin).

Hydrolysis of Triglycerides

Hydrolysis of pure triglycerides (TGs), including trilaurin, tripalmitinand tristearin, was carried out. A high amount of FFA was producedduring the reaction and high amount of unreacted TG remained in thereaction mixture. Most DG was 1,2-DG. Hydrolysis reaction showed higheracyl migration than alcoholysis reaction.

Effect of amount of water: highest yield was obtained using TG:waterratio of 1:1, as illustrated in FIG. 20. FIG. 20 shows the effect oftrilaurin:water ratio on hydrolysis of trilaurin in MTBE at 60° C. byLipozyme RM IM™ (DG=1,2-dilaurin+1,3-dilaurin, MG=monolaurin). Theamount of MG was increased with increasing TG:water ratio, especially inMTBE.

Effect of solvents: the result was corresponding to the result obtainedfrom alcoholysis reaction. Hydrolysis in MTBE allowed higher yield thanin n-hexane, isooctane and acetone, respectively, as illustrated in FIG.21. FIG. 21 summarizes data showing the effect of organic solvents onhydrolysis of trilaurin at 60° C. using Lipozyme RM IM™(DG=1,2-dilaurin+1,3-dilaurin, MG=1-monolaurin+2-monolaurin). Thoughreaction in MTBE gave higher yield, high amount of FFA was produced andlower acyl migration was found than in n-hexane. The separation of TG,DG, MG and FFA can be a problem.

Alcoholysis and Hydrolysis of Natural Oils

Alcoholysis and hydrolysis of natural oils, including coconut and palmkernel oils, was carried out. The 1,3-DG yield of reaction with naturaloils was slightly less than the yield of alcoholysis and hydrolysis ofpure TG. The highest DG yield (45-50%) and fastest reaction rate wasobtained from alcoholysis in MTBE at 40° C. by D-EP100, as illustratedin FIG. 22. FIG. 22 shows the results of alcoholysis and hydrolysis ofcoconut oil in organic solvent at 40° C. (TG:ethanol=1:1 mol/mol,TG:water=1:2 mol/mol). Lipozyme RM IM™ gave higher yield and less MGthan Lipozyme TL™. Reaction performed in MTBE gave higher yield andfaster reaction rate than in n-hexane and acetone, respectively.

Induction of Acyl Migration

Acyl migration was carried out on natural oils, including coconut andpalm kernel oils. The effect of temperature and carrier was not clear.Almost no acyl migration was observed after 72 h. Addition of oleic acidto the reaction mixture slightly induced acyl migration, as illustratedin FIG. 23. FIG. 23 shows the effect of oleic acid on acyl migration of1,2-dipalmitin in n-hexane at room temperature. The acyl migration ratewas increased with increasing oleic acid:1,2-DP ratio.

Anion exchangers showed high induction of acyl migration, while cationexchange showed no effect. The acyl migration rate was increased withincreasing amount of anion exchanger, as illustrated in FIG. 24. FIG. 24shows the effect of the amount of anion exchanger on acyl migration of1,2-dipalmitin (5 mg/ml) in n-hexane at room temperature. A large amountof anion-exchanger was required to induce a fast acyl migration.

Esterification of 1,3-DG and FFA/FAVE

Esterification of 1,3-DLa and OA in n-hexane was carried out withimmobilized lipase from Pseudomonas sp. (Aman PS-D), Candida antarcticatype A (CAL-A), and Penicillium cyclopium (Lipase G). Molecular sievewas added to the reaction mixtures to remove the produced water. It wasfound that only PS-D was capable of catalyzing the esterificationreaction of 1,3-DG and oleic acid (OA) or vinyl oleate (OAVE). Thereaction was fast. Almost all 1,3-DG was consumed after 2 h for 1,3-DCyand 8 h for 1,3-DLa.

Table 1 shows the fatty acid compositions of the ST products thusobtained. By-products of CyOO and OOO were present due to thenon-specificity of PS-D, as illustrated in FIG. 25.

TABLE 1 Fatty acid composition of structured triglycerides products.Fatty acids (%)* Structured triglycerides C8:0 C12:0 C18:1 CyOCy 61.0 —39.0 LaOLa — 63.3 36.7 CyOCy (larger scale) 66.9 — 33.1 *determined byGC analysis

FIG. 25 shows data from the esterification in larger scale of1,3-dicaprylin and oleic acid vinyl ester in n-hexane at 60° C. by theimmobilized lipase from a Pseudomonas sp. (PS-D). Acylglycerolcomposition was analyzed by HPLC.

Vinyl oleate (OAVE) allowed much faster reaction than OA and 1,3-DCyallowed faster reaction than 1,3-DLa, as illustrated in FIG. 26. FIG. 26shows data 20 from the esterification of 1,3-DG and oleic acid (C18:1)or oleic acid vinyl ester (OAVE) in n-hexane at 60° C. using PS-D.Acylglycerol composition was determined by TLC/FID (Cy=reaction with1,3-dicaprylin, La=reaction with 1,3-dilaurin, TG=triglycerides,DG=1,3-diglycerides, VE=vinyl ester).

Example 8 Protease Activity Assays

The following example describes exemplary protease activity assays todetermine the catalytic activity of a protease (e.g., an enzyme of theinvention). These exemplary assays can be used to determine if apolypeptide (e.g., a protease) is within the scope of the invention.

The activity assays used for proteinases (active on proteins) includezymograms and liquid substrate enzyme assays. Three different types ofzymograms were used to measure activity: casein, gelatin and zein. Forthe liquid substrate enzyme assays, three main types were used: gelelectrophoresis, O-pthaldialdehyde (OPA), and fluorescent end pointassays. For both the gel electrophoresis and OPA assays, four differentsubstrates were used: zein, Soybean Trypsin Inhibitor (SBTI,SIGMA-Aldrich, T6522), wheat germ lectin and soybean lectin. Thesubstrate for the fluorescent end point assay was gelatin.

The activity assays used for proteinases and peptidases (active onpeptides) used pNA linked small peptide substrates. The assays includedspecificity end point assays, unit definition kinetic assays and pHassays.

The following example describes the above-mentioned exemplary proteaseactivity assays. These exemplary assays can be used to determine if apolypeptide is within the scope of the invention.

Protein (Proteinase Activity)

Casein Zymogram Gel Assays

Casein zymogram gels were used to assess proteinase activity. Theprotease activity assays were assessed using 4-16% gradient gels(Invitrogen Corp., Carlsbad, Calif.) containing casein conjugated to ablue dye and embedded within the gel matrix. All zymogram gels wereprocessed according to the manufacturer's instructions. Briefly, eachsample was mixed with an equal volume of 2× loading dye and incubatedwithout heating for ten minutes before loading. After electrophoresis,gels were incubated in a renaturing buffer to remove the SDS and allowthe proteins to regain their native form. Gels were then transferred toa developing solution and incubated at 37° C. for 4 to 24 hours. If aprotease digests the casein in the gel, a clear zone is produced againstthe otherwise blue background that corresponds to the location of theprotease in the gel. Negative controls (indicated with NC on gel images)were processed along with the experimental samples in each experimentand electrophoresed on the casein zymograms next to their correspondingprotease(s).

Unlike traditional SDS-PAGE, samples are not heat denatured prior toelectrophoresis of casein zymograms. As a result, it is sometimesdifficult to accurately assess the molecular weight of the proteases.For example, Subtilisin A (Sigma, P5380, indicated with Subt.A on thegel images), which was used as a positive control in these experiments,is predicted to be approximately 27 kDa in size. However, whenelectrophoresed through casein zymograms using the conditions described,Subtilisin A barely migrates into the gel and is visible only above 183kDa. Therefore, the zymograms do not define the MW of the proteasesindicated, but rather used as an indicator of activity.

Gelatin Zymogram Assays

Gelatin zymograms, Novex® Zymogram Gels, were performed according tomanufacturer's instructions (Invitrogen Corp., Carlsbad, Calif.). Unlikethe casein zymograms, gelatin zymograms were post-stained followingdevelopment using either a Colloidal Blue Staining Kit or theSIMPLYBLUE™ Safestain, (both from Invitrogen). Areas of proteaseactivity appeared as clear bands against a dark background.

Corn Zein Assays

Corn zein was used as substrate for protease activity assays, usingpowder, Z-3625 (Sigma Chemical Co. St. Louis, Mo.), and Aquazein, 10%solution (Freeman Industries, Tuckahoe, N.Y.). When fractionated througha SDS-PAGE gel, zein from both suppliers produced bands of 24 and 22kDa. The two zein bands correspond in molecular weight to thosepreviously described for alpha-zein, the most abundant subclass ofzeins, which are estimated to comprise 71-84% of total zein in corn(see, e.g., Consoli (2001) Electrophoresis 22:2983-2989).

Lyophilized culture supernatants containing active protease wereresuspended, dialyzed, and incubated with zein in 50 mM KPO₄, pH 7.5.Reactions were run in a 96-well microtiter format. “Substrate only” and“enzyme preparation only” controls were processed as well asexperimental samples. After 24 hours at 30° C., aliquots were removedand subjected to OPA, SDS-PAGE, or Zymogram analysis. In some cases,fresh aliquots were removed and analyzed after 48 or 72 hours at 30° C.Zein Zymogram: Aquazein was added to a final concentration of 0.075% ina 10% polyacrylamide gel. Aliquots of dialyzed protease samples wereelectrophoresed through the zein zymogram using standard conditions.Following electrophoresis, the zymogram gel was washed, incubated in arenaturing buffer, incubated overnight in a developing buffer optimizedfor protease activity (contains NaCl, CaCl₂, and Brij 35, in Tris bufferpH 8), and stained with Coomassie blue stain.

SDS-PAGE: Aliquots of equal volume were removed from each sample andsubjected to SDS-PAGE analysis. Following electrophoresis, proteins inthe gels were stained with SYPRO Orange (Molecular Probes) andvisualized using UV transillumination.

OPA: In the presence of Beta-mercaptoethanol (BME), OPA reacts with freeamino ends to produce a fluorescent imidazole that can be detected usinga standard fluorescence plate reader. In this assay, aliquots of equalvolume were removed from each sample and placed in a black fluorescenceplate. Samples were then diluted 1:10 in OPA reagents. Fluorescence(Ex=340 nm, Em=450 nm) was determined after a 5-minute incubation.

Soybean Trypsin Inhibitor Assays

Soybean Trypsin Inhibitor (SBTI, SIGMA-Aldrich, T6522) was used as asubstrate for protease activity. Lyophilized culture supernatantscontaining active protease were resuspended, dialyzed, and incubatedwith SBTI (1 mg/ml final conc.) at 37° C. in 50 mM KPO₄, pH 7.5.Substrate alone and enzyme preparation alone controls were processedalong with experimental samples. After 24 hours, aliquots were removedand subjected to OPA and SDS-PAGE analysis. SDS-PAGE: for SBTI,following electrophoresis, proteins in the gels were stained withCoomassie blue.

Wheat Germ Lectin Assays

Wheat germ lectin (WGA, EY Laboratories, L-2101, Pure) was used as asubstrate for protease activity. Lyophilized culture supernatantscontaining active protease were resuspended, dialyzed, and incubatedwith WGA (1 mg/ml final concentration) at 37° C. in 50 mM KPO₄, pH 7.5.Substrate alone and enzyme preparation alone controls were processedalong with experimental samples. After 24 hours, aliquots were removedand subjected to OPA and SDS-PAGE analysis as. SDS-PAGE: for WGA,following electrophoresis, proteins in the gels were stained withCoomassie blue.

Soybean Lectin Assays

Soybean lectin (SBA, EY Laboratories, L-1300, Crude) was used as asubstrate for protease activity. Lyophilized culture supernatantscontaining active protease were resuspended, dialyzed, and incubatedwith SBA (1 mg/ml final concentration) at 37° C. in 50 mM KPO₄, pH 7.5.Substrate alone and enzyme preparation alone controls were processedalong with experimental samples. After 24 hours, aliquots were removedand subjected to OPA and SDS-PAGE analysis. SDS-PAGE: for SBA, followingelectrophoresis, proteins in the gels were stained with Coomassie blue.

Gelatin in Fluorescent Liquid End Point Assay

DQ Gelatin (Molecular Probes, fluorescein conjugate, D-12054) was usedto assess the proteolytic activity of the proteases of the invention. DQgelatin is a protein that is so heavily labeled with a fluorophore thatits fluorescence is quenched when the molecule is intact. Proteases thatcleave the substrate will release the fluorophores from internalquenching and fluorescence will increase in proportion to the proteaseactivity. DQ Gelatin was diluted to a final concentration of 25 ug/ml in100 ul reactions containing a suitable buffer such as zymogramdeveloping buffer (Invitrogen) and varying amounts of proteasepreparations. Reactions were incubated in a 384 well, clear, flat-bottommicrotiter plate at 37° C. for various time periods from 1 hr toovernight. Fluorescence was monitored using a fluorescence plate readerafter incubation at 37° C. for various times.

Example 9 Simulation of PLC-Mediated Degumming

This example describes an exemplary use of a hydrolase of the invention,a phospholipase of the invention, comprising the simulation ofphospholipase C (PLC)-mediated degumming

Due to its poor solubility in water phosphatidylcholine (PC) wasoriginally dissolved in ethanol (100 mg/ml). For initial testing, astock solution of PC in 50 mM 3-morpholinopropanesulpholic acid or 60 mMcitric acid/NaOH at pH 6 was prepared. The PC stock solution (10 ul, 1μg/ul) was added to 500 μl of refined soybean oil (2% water) in anEppendorf tube. To generate an emulsion the content of the tube wasmixed for 3 min by vortexing. The oil and the water phase were separatedby centrifugation for 1 min at 13,000 rpm. The reaction tubes werepre-incubated at the desired temperature (37° C., 50° C., or 60° C.) and3 μl of PLC from Bacillus cereus (0.9 U/μl) were added to the waterphase. The disappearance of PC was analyzed by TLC usingchloroform/methanol/water (65:25:4) as a solvent system (see, e.g.,Taguchi (1975) supra) and was visualized after exposure to I₂ vapor. Theoil and water phases are separated after centrifugation and PLC is addedto the water phase, which contains the precipitated phosphatides(“gums”). The PLC hydrolysis takes place in the water phase. The timecourse of the reaction is monitored by withdrawing aliquots from thewater phase and analyzing them by TLC.

Example 10 Expression of Hydrolases (e.g., Phospholipases) of theInvention

This example describes the construction of a commercial productionstrain of the invention that can express multiple hydrolases of theinvention, e.g., phospholipase enzymes of the invention. In order toproduce a multi-enzyme formulation suitable for use in the degumming offood-grade vegetable oils (including soybean, canola, and sunflower), arecombinant expression strain can be generated that expresses twodifferent hydrolases of the invention, e.g., phospholipase enzymes ofthe invention, in the same expression host. For example, this strain maybe constructed to contain one or more copies of a hydrolase (e.g., aPLC) gene and one or more copies of another hydrolase gene (e.g., aphosphatidylinositol-PLC gene). These genes may exist on one plasmid,multiple plasmids, or the genes may be inserted into the genome of theexpression host by homologous recombination. When the genes areintroduced by homologous recombination, the genes may be introduced intoa single site in the host genome as a DNA expression cassette thatcontains one or more copies of both genes. Alternatively, one or morecopies of each gene may be introduced into distinct sites in the hostchromosome. The expression of these two gene sequences could be drivenby one type of promoter or each gene sequence may be driven by anindependent promoter. Depending on the number of copies of each gene andthe type of promoter, the final strain will express varying ratios ofeach active enzyme type. The expression strains can be constructed usingany Streptomyces or Bacillus, Bacillus cereus, E. coli, S. pombe, P.pastoris, or other gram-negative, gram-positive, or yeast expressionsystems.

In one aspect, the invention provides a two-enzyme system for degummingof soybean oil, wherein at least one enzyme is a hydrolase enzyme of theinvention. PLC plus PI-PLC produces more DAG than either enzyme alone.However both enzymes produce more DAG than a no enzyme control sample.In one aspect, reaction conditions comprise 1 milliliter soybean oil,˜0.4% initial moisture, 50° C., 0.2% Citric acid neutralized with 2.75MNaOH, 10U PLC, 15 μL PI-PLC (0.45 mg total protein), 1 hour totalreaction time. FIG. 31 illustrates a table summarizing data from thistwo-enzyme degumming system of the invention.

In another aspect, a PI-PLC enzyme of the invention can be used underthe same conditions described for PLC. These include chemical refiningof vegetable oils and water degumming of vegetable oils.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A nucleic acid that encodes a polypeptide having a hydrolase activityselected from the group consisting of: a lipase activity and aphospholipase activity, wherein the nucleic acid is a sequence having atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to SEQ ID NO:15.
 2. An expressioncassette, a vector, or a cloning vehicle comprising the nucleic acidsequence as set forth in claim
 1. 3. A transformed non-human cellcomprising the expression cassette as set forth in claim 2, wherein thetransformed non-human cell is a bacterial cell, a mammalian cell, afungal cell, a yeast cell, an insect cell, or a plant cell.
 4. Anisolated or recombinant polypeptide having hydrolase activity consistingof: a lipase activity, and a phospholipase activity wherein thepolypeptide is selected from: (i) an amino acid sequence having at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NO:16, or, (ii) encoded by thenucleic acid as set forth in claim
 1. 5. A protein preparationcomprising a polypeptide as set forth in claim 4, wherein the proteinpreparation comprises a liquid, a solid, or a gel.
 6. A food supplementfor an animal comprising the polypeptide as set forth in claim
 4. 7. Amethod of producing the recombinant polypeptide of claim 4 comprising:(a) providing the nucleic acid of claim 1 operably linked to a promoter;and (b) expressing the nucleic acid of step (a) under conditions thatallow expression of the polypeptide, thereby producing the recombinantpolypeptide of claim
 4. 8. A method for hydrolyzing a triacylglycerol(TAG), a diacylglycerol (DAG) or a monoacylglycerol (MAG) comprising thefollowing steps: (a) providing the polypeptide as set forth in claim 4;(b) providing a composition comprising a triacylglycerol (TAG), adiacylglycerol (DAG) or a monoacylglycerol (MAG); and (c) contacting thepolypeptide of step (a) with the composition of step (b) underconditions wherein the polypeptide hydrolyzes the triacylglycerol (TAG),diacylglycerol (DAG), or monoacylglycerol (MAG).
 9. A detergentcomposition comprising the polypeptide as set forth in claim
 4. 10. Thedetergent composition of claim 9, wherein the polyeptide is anonsurface-active hydrolase or a surface-active hydrolase.
 11. Thedetergent composition of claim 9, wherein the polypeptide is formulatedin a non-aqueous liquid composition, a cast solid, a granular form, aparticulate form, a compressed tablet, a gel form, a paste, or a slurryform.
 12. A method for refining a lubricant comprising the followingsteps: (a) providing the polypeptide as set forth in claim 4; (b)providing a lubricant; and (c) treating the lubricant with thepolypeptide of (a) under conditions wherein the polypeptide canselective hydrolyze oils in the lubricant, thereby refining it.
 13. Amethod of treating a fabric comprising the following steps: (a)providing the polypeptide as set forth in claim 4; (b) providing afabric; and (c) treating the fabric with the polypeptide of (a) undercondition wherein the polypeptide can selectively hydrolyze carboxylicesters thereby treating the fabric.
 14. The method of claim 13, whereintreatment of the fabric comprises improvement of the hand and drape ofthe final fabric, dyeing, obtaining flame retardancy, obtaining waterrepellency, obtaining optical brightness, or obtaining resin finishing.15. A method for removing or decreasing the amount of a food or oilstain comprising contacting the polypeptide of claim 4 with the food oroil stain under conditions wherein the polypeptide can hydrolyze food oroil stain.
 16. The method of claim 15, wherein the polypeptide of claim4 has an enhanced stability to denaturation by surfactants and to heatdeactivation.
 17. A dietary composition comprising the polypeptide asset forth in claim
 4. 18. A method of reducing fat content in milk orvegetable-based dietary compositions comprising the following steps: (a)providing the polypeptide as set forth in claim 4; (b) providing acomposition comprising a milk or a vegetable oil, and (c) treating thecomposition of step (b) with the polypeptide of (a) under conditionswherein the polypeptide can hydrolyze the oil or fat in the compositionof (b), thereby reducing its fat content.
 19. A method of catalyzing aninteresterification or transesterification reaction to produce newtriglycerides comprising the following steps: (a) providing thepolypeptide of claim 4, wherein the polypeptide can catalyze aninteresterification or transterification reaction; (b) providing amixture of triglycerides (TAG), a diacylglycerol (DAG), amonoacylglycerol (MAG) or free fatty acids; (c) treating the compositionof step (b) with the polypeptide of (a) under conditions wherein thepolypeptide can catalyze exchange of free fatty acids with the acylgroups of triglycerides(TAG), the diacylglycerol (DAG), themonoacylglycerol (MAG), thereby producing new triglycerides enriched inthe added fatty acids.
 20. A method for ameliorating or preventinglipopolysaccharide (LPS)-mediated toxicity comprising administering to apatient a pharmaceutical composition comprising the polypeptide as setforth in claim
 4. 21. A method for detoxifying an endotoxin comprisingcontacting the endotoxin with the polypeptide as set forth in claim 4.